Optimized variants of anti-vegf antibodies

ABSTRACT

The present invention provides anti-VEGF antibodies and compositions that include anti-VEGF antibodies (e.g., antibody conjugates, fusion proteins, and polymeric formulations), and uses thereof, for example for treatment of disorders associated with pathological angiogenesis. The present invention also provides methods of identifying antibody variants with improved properties, for example, enhanced binding affinity, stability, pharmacokinetics, and/or expression.

FIELD OF THE INVENTION

The invention relates generally to anti-VEGF antibodies, as well ascompositions that include anti-VEGF antibodies (e.g., antibodyconjugates, fusion proteins, and polymeric formulations), withbeneficial properties for research, therapeutic, and diagnosticpurposes. The invention also relates to methods of identifying antibodyvariants with improved properties, for example, enhanced bindingaffinity, stability, and/or expression.

BACKGROUND OF THE INVENTION

Angiogenesis is a tightly-regulated process through which new bloodvessels form from pre-existing blood vessels. Although angiogenesis isimportant during development to ensure adequate blood circulation, manydisorders are associated with pathological angiogenesis, such as oculardisorders (e.g., age-related macular degeneration, AMD) and cellproliferative disorders (e.g., cancer). Vascular endothelial growthfactor (VEGF) is a clinically-validated driver of angiogenesis andneutralization of VEGF, for example, using an anti-VEGF blockingantibody, can be used to treat disorders associated with pathologicalangiogenesis.

There remains a need for antibodies, such as anti-VEGF antibodies, withenhanced binding affinity, stability, pharmacokinetics, and/orexpression, for example, for use in treating disorders associated withpathological angiogenesis. In particular, there is a need for antibodycompositions for long-acting delivery for treatment of ocular disorders(e.g., AMD (e.g., wet AMD), diabetic macular edema (DME), diabeticretinopathy (DR), and retinal vein occlusion (RVO)). In addition, thereis an unmet need for improved methods of identifying such antibodieswith improved properties (e.g., enhanced binding affinity, stability,pharmacokinetics, and/or expression).

SUMMARY OF THE INVENTION

The present invention provides anti-VEGF antibodies, compositions thatinclude anti-VEGF antibodies (e.g., antibody conjugates, fusionproteins, and polymeric formulations), and methods of use thereof, forexample, for treatment of disorders associated with pathologicalangiogenesis (e.g., ocular disorders and cell proliferative disorders).The present invention also provides methods of identifying antibodyvariants with improved properties, for example, enhanced bindingaffinity, stability, pharmacokinetics, and/or expression.

In one aspect, the invention features an isolated antibody thatspecifically binds vascular endothelial growth factor (VEGF), whereinthe antibody comprises the following six hypervariable regions (HVRs):(a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO:1); (b) an HVR-H2 comprising the amino acid sequence ofGX₁TPX₂GGX₃X₄X₅YX₆DSVX₇X₈ (SEQ ID NO: 2), wherein X₁ is lie or His, X₂is Ala or Arg, X₃ is Tyr or Lys, X₄ is Thr or Glu, X₅ is Arg, Tyr, Gln,or Glu, X₆ is Ala or Glu, X₇ is Lys or Glu, and X₈ is Gly or Glu; (c) anHVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3);(d) an HVR-L1 comprising the amino acid sequence of RASQX₁VSTAVA (SEQ IDNO: 4), wherein X₁ is Asp or Arg; (e) an HVR-L2 comprising the aminoacid sequence of X₁ASFLYS (SEQ ID NO: 5), wherein X₁ is Ser or Met; and(f) an HVR-L3 comprising the amino acid sequence of X₁QGYGX₂PFT (SEQ IDNO: 6), wherein X₁ is Gln, Asn, or Thr and X₂ is Ala, Asn, Gln, or Arg.In some embodiments, the antibody comprises the following six HVRs: (a)an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1);(b) an HVR-H2 comprising the amino acid sequence of GITPAGGYTRYADSVKG(SEQ ID NO: 7), GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG(SEQ ID NO: 22); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10) orQQGYGNPFT (SEQ ID NO: 23).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some embodiments, the antibody further comprises the following heavychain variable (VH) domain framework regions (FRs): (a) an FR-H1comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS(SEQ ID NO: 13); (b) an FR-H2 comprising the amino acid sequence ofWVRQAPGKGLEWVA (SEQ ID NO: 14); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).In some embodiments, the antibody further comprises the following lightchain variable (VL) domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some embodiments, the antibody further comprises the following VLdomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 24); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some embodiments, the antibody further comprises the following VLdomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome embodiments, the antibody further comprises the following VH domainFRs: (a) an FR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 11, 40, or 42; (b) a VL domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 12, 41, or 46; or (c) a VHdomain as in (a) and a VL domain as in (b). In some embodiments, the VHdomain further comprises the following FRs: (a) an FR-H1 comprising theamino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13);(b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQID NO: 14) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprisingthe amino acid sequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO:15); and (d) an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS(SEQ ID NO: 16). In some embodiments, the VH domain comprises the aminoacid sequence of SEQ ID NO: 11. In some embodiments, the VL domainfurther comprises the following FRs: (a) an FR-L1 comprising the aminoacid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) orDIQMTQSPSSLSASVGDRVTIDC (SEQ ID NO: 45); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), orGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20) orFGQGTKVEVK (SEQ ID NO: 55). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO:12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 11 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 40 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 42 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 42 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 41.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 33 or 51; (b) a VL domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 12, 34, 35, 36, 37, or 38; or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following FRs: (a) anFR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 33. In some embodiments,the VH domain comprises the amino acid sequence of SEQ ID NO: 51. Insome embodiments, the antibody further comprises the following FRs: (a)an FR-L1 comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), orDIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome embodiments, the VL domain comprises the amino acid sequence of SEQID NO: 34. In some embodiments, the VL domain comprises the amino acidsequence of SEQ ID NO: 35. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 36. In some embodiments, the VLdomain comprises the amino acid sequence of SEQ ID NO: 37. In someembodiments, the VL domain comprises the amino acid sequence of SEQ IDNO: 12. In some embodiments, the VL domain comprises the amino acidsequence of SEQ ID NO: 38.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 38.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 34.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 35.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 36.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 37.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 38.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 35.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 37.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 48 and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 50.

In another aspect, the invention features an isolated antibody thatspecifically binds VEGF, wherein the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 49 and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of any of the preceding aspects, the antibody iscapable of inhibiting the binding of VEGF to a VEGF receptor. In someembodiments, the VEGF receptor is VEGF receptor 1 (Flt-1). In someembodiments, the VEGF receptor is VEGF receptor 2 (KDR).

In some embodiments of any of the preceding aspects, the antibody bindshuman VEGF (hVEGF) with a Kd of about 2 nM or lower. In someembodiments, the antibody binds hVEGF with a Kd between about 75 pM andabout 2 nM. In some embodiments, the antibody binds hVEGF with a Kdbetween about 75 pM and about 600 pM. In some embodiments, the antibodybinds hVEGF with a Kd between about 75 pM and about 500 pM. In someembodiments, the antibody binds hVEGF with a Kd of about 80 pM. In someembodiments, the antibody binds hVEGF with a Kd of about 60 pM.

In some embodiments of any of the preceding aspects, the antibody has amelting temperature (Tm) of greater than about 83.5° C. In someembodiments, the antibody has a Tm of about 85° C. to about 91° C. Insome embodiments, the antibody has a Tm of about 89° C.

In some embodiments of any of the preceding aspects, the antibody has anisoelectric point (pI) of lower than 8. In some embodiments, theantibody has a pI from about 5 to about 7. In some embodiments, theantibody has a pI of from about 5 to about 6.

In some embodiments of any of the preceding aspects, the antibody ismonoclonal, human, humanized, or chimeric.

In some embodiments of any of the preceding aspects, the antibody is anantibody fragment that binds VEGF. In some embodiments, the antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments. In some embodiments, the antibodyfragment is an Fab.

In some embodiments of any of the preceding aspects, the antibody is amonospecific antibody. In other embodiments of any of the precedingaspects, the antibody is a multispecific antibody. In some embodiments,the multispecific antibody is a bispecific antibody. In someembodiments, the bispecific antibody binds VEGF and a second biologicalmolecule selected from the group consisting of interleukin 1β (IL-1β);interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13(IL-13); IL-13 receptor (IL-13R); PDGF; angiopoietin; angiopoietin 2;Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and a protein genetically linked to age-related maculardegeneration (AMD) risk. In some embodiments, the VEGF receptor isVEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF-receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR). In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1;TLR3; TLR4; CETP; LIPC, COL10A1; and TNFRSF10A.

In another aspect, the invention features a polynucleotide (e.g., anisolated polynucleotide) encoding any of the antibodies describedherein. In another aspect, the invention features a vector (e.g., anexpression vector) comprising the polynucleotide for expressing theantibody. In another aspect, the invention features host cellscomprising the preceding polynucleotides and/or vectors. In someembodiments, the host cell is a mammalian cell. In some embodiments, themammalian cell is a 293 cell, a Chinese hamster ovary (CHO) cell, ayeast cell, or a plant cell. In some embodiments, the host cell is aprokaryotic cell. In some embodiments, the prokaryotic cell is E. coli.

In another aspect, the invention features a method of producing any ofthe antibodies described herein, the method comprising culturing a hostcell that comprises any of the preceding vectors (e.g., expressionvectors) in a culture medium. In some embodiments, the method furthercomprises recovering the antibody from the host cell or the culturemedium. In some embodiments, the mammalian cell is a 293 cell, a Chinesehamster ovary (CHO) cell, a yeast cell, or a plant cell. In someembodiments, the host cell is a prokaryotic cell. In some embodiments,the prokaryotic cell is E. coli.

In another aspect, the invention features a method of reducing orinhibiting angiogenesis in a subject having a disorder associated withpathological angiogenesis, comprising administering to the subject aneffective amount of any one of the preceding antibodies, therebyreducing or inhibiting angiogenesis in the subject. In some embodiments,the disorder associated with pathological angiogenesis is an oculardisorder or a cell proliferative disorder. In some embodiments, thedisorder associated with pathological angiogenesis is an oculardisorder. In some embodiments, the ocular disorder is selected from thegroup consisting of age-related macular degeneration (AMD), maculardegeneration, macular edema, diabetic macular edema (DME) (includingfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (including proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, retinopathy of prematurity (ROP),retinal vein occlusion (RVO) (including central (CRVO) and branched(BRVO) forms), CNV (including myopic CNV), corneal neovascularization, adisease associated with corneal neovascularization, retinalneovascularization, a disease associated with retinal/choroidalneovascularization, pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR),Coats' disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome(OPPG), subconjunctival hemorrhage, rubeosis, ocular neovasculardisease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, conjunctivitis (including infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, and Sjögren's disease. In some embodiments, the ocular disorderis AMD, DME, DR, or RVO. In some embodiments, the ocular disorder isAMD. In some embodiments, the AMD is wet AMD. In some embodiments, thedisorder associated with pathological angiogenesis is a cellproliferative disorder. In some embodiments, the cell proliferativedisorder is a cancer. In some embodiments, the cancer is selected fromthe group consisting of breast cancer, colorectal cancer, non-small celllung cancer, non-Hodgkins lymphoma (NHL), renal cancer, prostate cancer,liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma.

In another aspect, the invention features a method for treating adisorder associated with pathological angiogenesis, the methodcomprising administering an effective amount of any one of the precedingantibodies to a subject in need of such treatment. In some embodiments,the disorder associated with pathological angiogenesis is an oculardisorder or a cell proliferative disorder. In some embodiments, thedisorder associated with pathological angiogenesis is an oculardisorder. In some embodiments, the ocular disorder is selected from thegroup consisting of age-related macular degeneration (AMD), maculardegeneration, macular edema, diabetic macular edema (DME) (includingfocal, non-center DME and diffuse. center-involved DME), retinopathy,diabetic retinopathy (DR) (including proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, retinopathy of prematurity (ROP),retinal vein occlusion (RVO) (including central (CRVO) and branched(BRVO) forms), CNV (including myopic CNV), corneal neovascularization, adisease associated with corneal neovascularization, retinalneovascularization, a disease associated with retinal/choroidalneovascularization, pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR),Coats' disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome(OPPG), subconjunctival hemorrhage, rubeosis, ocular neovasculardisease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, conjunctivitis (including infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, and Sjögren's disease. In some embodiments, the ocular disorderis AMD, DME, DR, or RVO. In some embodiments, the ocular disorder isAMD. In some embodiments, the AMD is wet AMD. In some embodiments, thedisorder associated with pathological angiogenesis is a cellproliferative disorder. In some embodiments, the cell proliferativedisorder is a cancer. In some embodiments, the cancer is selected fromthe group consisting of breast cancer, colorectal cancer, non-small celllung cancer, non-Hodgkins lymphoma (NHL), renal cancer, prostate cancer,liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma.

In another aspect, the invention features a method of inhibitingvascular permeability in a subject suffering from a disorder associatedwith undesirable vascular permeability, the method comprisingadministering to the subject an effective amount of any one of thepreceding antibodies, thereby inhibiting vascular permeability in thesubject.

In another aspect, the invention features a method of treating adisorder associated with undesirable vascular permeability, the methodcomprising administering an effective amount of any one of the precedingantibodies to a subject in need of such treatment.

In some embodiments of any of the preceding aspects, the disorderassociated with undesirable vascular permeability is selected from thegroup consisting of edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion, pleural effusion, andpermeability associated with cardiovascular diseases.

In some embodiments of any of the preceding aspects, the method furthercomprises administering to the subject an effective amount of a secondagent, wherein the second agent is selected from the group consisting ofanother antibody, a chemotherapeutic agent, a cytotoxic agent, ananti-angiogenic agent, an immunosuppressive agent, a prodrug, acytokine, a cytokine antagonist, cytotoxic radiotherapy, acorticosteroid, an anti-emetic, a cancer vaccine, an analgesic, agrowth-inhibitory agent, and a compound that binds to a secondbiological molecule. In some embodiments, the anti-angiogenic agent is aVEGF antagonist. In some embodiments, the VEGF antagonist is ananti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin. or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTISS), RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab)₂ fragments.

In some embodiments of any of the preceding aspects, the antibody isadministered intravitreally, ocularly, intraocularly, juxtasclerally,subtenonly, superchoroidally, topically, intravenously, intramuscularly,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, transdermally, by inhalation,by injection, by eye drop, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. In someembodiments, the antibody is administered intravitreally, ocularly,intraocularly, juxtasclerally, subtenonly, superchoroidally, ortopically. In some embodiments, the antibody is administeredintravitreally by injection.

In some embodiments, the antibody is administered topically by eye dropor ointment. In some embodiments, the antibody is administered by a portdelivery device. In some embodiments, the subject is a human.

In another aspect, the invention features a pharmaceutical compositioncomprising any one of the preceding antibodies. In some embodiments, thepharmaceutical formulation further comprises a polymer.

In some embodiments, the polymer is a biodegradable polymer. In someembodiments, the pharmaceutical composition is formulated as a polymersolvent depot, a polymer implant, or a polymer micelle. In someembodiments, the polymer is a polylactic acid polyglycolic acid (PLGA)copolymer. In some embodiments, the pharmaceutical composition isformulated as a PLGA rod. In some embodiments, the pharmaceuticalcomposition is used for treating a disorder associated with pathologicalangiogenesis or a disorder associated with undesirable vascularpermeability in a mammal. In some embodiments, the disorder associatedwith pathological angiogenesis is an ocular disorder or a cellproliferative disorder. In some embodiments, the disorder associatedwith pathological angiogenesis is an ocular disorder. In someembodiments, the ocular disorder is selected from the group consistingof age-related macular degeneration (AMD), macular degeneration, macularedema, diabetic macular edema (DME) (including focal, non-center DME anddiffuse, center-involved DME), retinopathy, diabetic retinopathy (DR)(including proliferative DR (PDR), non-proliferative DR (NPDR), andhigh-altitude DR), other ischemia-related retinopathies, retinopathy ofprematurity (ROP), retinal vein occlusion (RVO) (including central(CRVO) and branched (BRVO) forms), CNV (including myopic CNV), cornealneovascularization, a disease associated with cornealneovascularization, retinal neovascularization, a disease associatedwith retinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, familial exudativevitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD. In some embodiments, the disorderassociated with pathological angiogenesis is a cell proliferativedisorder. In some embodiments, the cell proliferative disorder is acancer. In some embodiments, the cancer is selected from the groupconsisting of breast cancer, colorectal cancer, non-small cell lungcancer, non-Hodgkins lymphoma (NHL), renal cancer, prostate cancer,liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. In some embodiments, the disorderassociated with undesirable vascular permeability is selected from thegroup consisting of edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion, pleural effusion, andpermeability associated with cardiovascular diseases. In someembodiments, the pharmaceutical composition further comprises a secondagent, wherein the second agent is selected from the group consisting ofanother antibody, a chemotherapeutic agent, a cytotoxic agent, ananti-angiogenic agent, an immunosuppressive agent, a prodrug, acytokine, a cytokine antagonist, cytotoxic radiotherapy, acorticosteroid, an anti-emetic, a cancer vaccine, an analgesic, agrowth-inhibitory agent, and a compound that binds to a secondbiological molecule. In some embodiments, the anti-angiogenic agent is aVEGF antagonist. In some embodiments, the VEGF antagonist is ananti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin®, or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab)₂ fragments.

In another aspect, the invention features an antibody conjugatecomprising (i) any one of the preceding antibodies and (ii) ahydrophilic polymer covalently attached to the antibody. In someembodiments, the hydrophilic polymer is a hyaluronic acid (HA) polymeror a polyethylene glycol (PEG) polymer. In some embodiments, thehydrophilic polymer is an HA polymer. In some embodiments, the HApolymer has a molecular weight of about 1 megadalton (MDa) or lower. Insome embodiments of any of the preceding aspects, the HA polymer has amolecular weight between about 25 kDa and about 500 kDa. In someembodiments, the HA polymer has a molecular weight between about 100 kDaand about 250 kDa. In some embodiments, the HA polymer has a molecularweight of about 200 kDa. In some embodiments, the HA polymer is notcross-linked. In some embodiments, the antibody is an antibody fragmentthat binds VEGF. In some embodiments, the antibody fragment is selectedfrom the group consisting of Fab, Fab-C, Fab′, Fab′-SH, Fv, scFv, and(Fab)₂ fragments. In some embodiments, the antibody fragment is an Fab,Fab-C, or Fab′. In some embodiments, the antibody conjugate has ahydrodynamic radius between about 10 nm and about 60 nm. In someembodiments, the antibody conjugate has a hydrodynamic radius betweenabout 25 nm and about 35 nm. In some embodiments, the hydrodynamicradius is about 28 nm. In some embodiments, the antibody conjugate hasan ocular half-life that is increased relative to a reference antibodythat is not covalently attached to the hydrophilic polymer. In someembodiments, the ocular half-life is increased at least about 2-foldrelative to the reference antibody. In some embodiments, the ocularhalf-life is increased at least about 4-fold relative to the referenceantibody. In some embodiments, the ocular half-life is a vitrealhalf-life. In some embodiments, the reference antibody is identical tothe antibody of the antibody conjugate.

In another aspect, the invention features an antibody conjugatecomprising (i) an antibody that 15 specifically binds VEGF and (ii) anHA polymer covalently attached to the antibody, wherein the HA polymerhas a molecular weight of 1 MDa or lower. In some embodiments of any ofthe preceding aspects, the HA polymer has a molecular weight betweenabout 25 kDa and about 500 kDa. In some embodiments, the HA polymer hasa molecular weight between about 100 kDa and about 250 kDa. In someembodiments, the HA polymer has a molecular weight of about 200 kDa. Insome embodiments, the HA polymer is not cross-linked. In someembodiments, the antibody is an antibody fragment that binds VEGF. Insome embodiments, the antibody fragment is selected from the groupconsisting of Fab, Fab-C, Fab′, Fab′-SH, Fv, scFv, and (Fab)₂ fragments.In some embodiments, the antibody fragment is an Fab, Fab-C, or Fab′. Insome embodiments, the antibody conjugate has a hydrodynamic radiusbetween about 10 nm and about 60 nm. In some embodiments, the antibodyconjugate has a hydrodynamic radius between about 25 nm and about 35 nm.In some embodiments, the hydrodynamic radius is about 28 nm. In someembodiments, the antibody conjugate has an ocular half-life that isincreased relative to a reference antibody that is not covalentlyattached to the hydrophilic polymer. In some embodiments, the ocularhalf-life is increased at least about 2-fold relative to the referenceantibody. In some embodiments, the ocular half-life is increased atleast about 4-fold relative to the reference antibody. In someembodiments, the ocular half-life is a vitreal half-life. In someembodiments, the reference antibody is identical to the antibody of theantibody conjugate.

In some embodiments of any of the preceding aspects, the antibody iscovalently attached to the polymer by a reversible prodrug linker. Insome embodiments, the polymer is a hydrogel. In some embodiments, thehydrogel is a PEG-based hydrogel. In some embodiments, the hydrogel isin the shape of a microparticulate bead.

In another aspect, the invention features a fusion protein comprisingany one of the preceding antibodies covalently attached to an HA bindingdomain. In some embodiments, the HA binding domain is covalentlyattached to the heavy chain or the light chain of the antibody. In someembodiments, the HA binding domain is covalently attached to theC-terminus of the heavy chain or to the C-terminus of the light chain.In some embodiments, the HA binding domain is covalently attached to theC-terminus of the heavy chain. In some embodiments, the HA bindingdomain is covalently attached to the C-terminus of the light chain. Insome embodiments, the fusion protein further comprises a linker, thelinker being positioned between the antibody and the HA binding domain.In some embodiments, the linker comprises the amino acid sequence ofGGGGS (SEQ ID NO: 61). In some embodiments, the linker consists of theamino acid sequence of GGGGS (SEQ ID NO: 61). In some embodiments, theantibody is an antibody fragment that binds VEGF. In some embodiments,the antibody fragment is selected from the group consisting of Fab,Fab-C, Fab′, Fab′-SH, Fv, scFv, and (Fab)₂ fragments. In someembodiments, the antibody fragment is an Fab. In some embodiments, theHA binding domain is covalently attached to the C-terminus of the CH1domain of the Fab. In some embodiments, the HA binding protein iscovalently attached to the C-terminus of the CL domain of the Fab. Insome embodiments, the HA binding domain is selected from the groupconsisting of a link module, a G1 domain, and a lysine-richoligopeptide. In some embodiments, the HA binding domain is a linkmodule. In some embodiments, the link module is selected from the groupconsisting of tumor necrosis factor-stimulated gene 6 (TSG6), CD44,lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), hyaluronanand proteoglycan link protein (HAPLN) 1, HAPLN2, HAPLN3, HAPLN4,aggrecan, brevican, neurocan, phosphacan, versican, CAB61358, KIA0527,stabilin-1, and stabilin-2 link modules. In some embodiments, the linkmodule is a TSG6 link module. In some embodiments, the TSG6 link moduleis a human TSG6 link module or a rabbit TSG6 link module. In someembodiments, the TSG link module is a human TSG6 link module. In someembodiments, the human TSG6 link module comprises amino acid residues36-128 of human TSG6.

In some embodiments of any of the preceding aspects, the fusion proteinfurther comprises at least one additional HA binding domain. In someembodiments, the at least one additional HA binding domain is covalentlyattached to the heavy chain or the light chain of the antibody. In someembodiments, the at least one additional HA binding protein is linked tothe antibody by a linker. In some embodiments, the linker comprises theamino acid sequence of GGGGS (SEQ ID NO: 61). In some embodiments, thelinker consists of the amino acid sequence of GGGGS (SEQ ID NO: 61). Insome embodiments, a first HA binding domain is covalently attached tothe heavy chain and a second HA binding domain is covalently attached tothe light chain. In some embodiments, the first HA binding domain islinked to the C-terminus of the heavy chain and the second HA bindingdomain is linked to the C-terminus of the light chain. In someembodiments, the at least one additional HA binding protein is selectedfrom the group consisting of a link module, a G1 domain, and alysine-rich oligopeptide. In some embodiments, the at least oneadditional HA binding protein is a link module. In some embodiments, thelink module is a TSG6 link module. In some embodiments, the TSG6 linkmodule is a human TSG6 link module or a rabbit TSG6 link module. In someembodiments, the TSG6 link module is a human TSG6 link module. In someembodiments, the human TSG6 link module comprises amino acid residues36-128 of human TSG6.

In some embodiments of any of the preceding aspects, the fusion proteinspecifically binds to VEGF and HA. In some embodiments, the fusionprotein binds HA with a Kd of about 2 μM or lower. In some embodiments,the fusion protein binds HA with a Kd between about 1 nM and about 500nM. In some embodiments, the fusion protein binds HA with a Kd betweenabout 1 nM and about 50 nM. In some embodiments, the fusion proteinbinds HA with a Kd of about 10 nM.

In some embodiments of any of the preceding aspects, the fusion proteinhas an ocular half-life that is increased relative to a referenceantibody that is not covalently attached to a HA binding domain. In someembodiments, the ocular half-life is increased at least about 2-foldrelative to the reference antibody. In some embodiments, the ocularhalf-life is increased at least about 4-fold relative to the referenceantibody. In some embodiments, the ocular half-life is a vitrealhalf-life. In some embodiments, the reference antibody is identical tothe antibody of the fusion protein.

In another aspect, the invention features a method of reducing orinhibiting angiogenesis in a subject having a disorder associated withpathological angiogenesis, comprising administering to the subject aneffective amount of any one of the preceding antibody conjugates,thereby reducing or inhibiting angiogenesis in the subject.

In another aspect, the invention features a method for treating adisorder associated with pathological angiogenesis, the methodcomprising administering an effective amount of any one of the precedingantibody conjugates to a subject in need of such treatment.

In another aspect, the invention features a method of reducing orinhibiting angiogenesis in a subject having a disorder associated withpathological angiogenesis, comprising administering to the subject aneffective amount of any one of the preceding fusion proteins, therebyreducing or inhibiting angiogenesis in the subject.

In another aspect, the invention features a method for treating adisorder associated with pathological angiogenesis, the methodcomprising administering an effective amount of any one of the precedingfusion proteins to a subject in need of such treatment.

In some embodiments of any of the preceding aspects, the disorderassociated with pathological angiogenesis is an ocular disorder. In someembodiments, the ocular disorder is selected from the group consistingof AMD, macular degeneration, macular edema, DME (including focal,non-center DME and diffuse, center-involved DME), retinopathy, DR(including PDR, NPDR, and high-altitude DR), other ischemia-relatedretinopathies, ROP, RVO (including CRVO and BRVO forms), CNV (includingmyopic CNV), corneal neovascularization, a disease associated withcorneal neovascularization, retinal neovascularization, a diseaseassociated with retinal/choroidal neovascularization, pathologic myopia,von Hippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats'disease, Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis,ocular neovascular disease, neovascular glaucoma, RP, hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, CME, vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD.

In some embodiments of any of the preceding aspects, the method furthercomprises administering to the subject an effective amount of a secondagent, wherein the second agent is selected from the group consisting ofanother antibody, an anti-angiogenic agent, a cytokine, a cytokineantagonist, a corticosteroid, an analgesic, and a compound that binds toa second biological molecule. In some embodiments, the anti-angiogenicagent is a VEGF antagonist. In some embodiments, the VEGF antagonist isan anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin®, or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTIS®). RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab)₂ fragments.

In some embodiments of any of the preceding aspects, the antibodyconjugate is administered intravitreally, ocularly, intraocularly,juxtasderally, subtenonly, superchoroidally, topically, intravenously,intramuscularly, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intrathecally,intranasally, intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, transdermally, by inhalation,by injection, by eye drop, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. In someembodiments, the antibody conjugate is administered intravitreally,ocularly, intraocularly, juxtasclerally, subtenonly, superchoroidally,or topically. In some embodiments, the antibody conjugate isadministered intravitreally by injection. In some embodiments, theantibody conjugate is administered topically by eye drop or ointment. Insome embodiments, the antibody conjugate is administered by a portdelivery device.

In some embodiments of any of the preceding aspects, the fusion proteinis administered intravitreally, ocularly, intraocularly, juxtasclerally,subtenonly, superchoroidally, topically, intravenously, intramuscularly,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, transdermally, by inhalation,by injection, by eye drop, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. In someembodiments, the fusion protein is administered intravitreally,ocularly, intraocularly, juxtasclerally, subtenonly, superchoroidally,or topically. In some embodiments, the fusion protein is administeredintravitreally by injection. In some embodiments, the fusion protein isadministered topically by eye drop or ointment. In some embodiments, thefusion protein is administered by a port delivery device. In someembodiments, the subject is a human.

In another aspect, the invention features a pharmaceutical compositioncomprising any one of the preceding antibody conjugates.

In another aspect, the invention features a pharmaceutical compositioncomprising any one of the preceding fusion proteins.

In some embodiments of any of the preceding aspects, the pharmaceuticalcomposition is used for treating a disorder associated with pathologicalangiogenesis in a mammal. In some embodiments, the disorder associatedwith pathological angiogenesis is an ocular disorder. In someembodiments, the ocular disorder is selected from the group consistingof AMD, macular degeneration, macular edema, DME (including focal,non-center DME and diffuse, center-involved DME), retinopathy, DR(including PDR, NPDR, and high-altitude DR), other ischemia-relatedretinopathies, ROP, RVO (including CRVO and BRVO forms), CNV (includingmyopic CNV), corneal neovascularization, a disease associated withcorneal neovascularization, retinal neovascularization, a diseaseassociated with retinal/choroidal neovascularization, pathologic myopia,von Hippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats'disease, Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis,ocular neovascular disease, neovascular glaucoma, RP, hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, CME, vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD.

In some embodiments of any of the preceding aspects, the pharmaceuticalcomposition further comprises a second agent, wherein the second agentis selected from the group consisting of another antibody, ananti-angiogenic agent, a cytokine, a cytokine antagonist, acorticosteroid, an analgesic, and a compound that binds to a secondbiological molecule. In some embodiments, the anti-angiogenic agent is aVEGF antagonist. In some embodiments, the VEGF antagonist is ananti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin®, or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(I-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab)₂ fragments.

In another aspect, the invention features a method of identifying anamino acid residue alteration that confers enhanced binding of anantibody to a target molecule, the method comprising: (a) providing adisplay library comprising nucleic acids encoding candidate antibodyvariants, wherein each candidate antibody variant comprises an aminoacid residue alteration in the VH or the VL compared to a referenceantibody, and wherein amino acid residue alterations at every positionof the VH or VL are present in the display library; (b) sorting thedisplay library based on binding of the candidate antibody variants tothe target molecule to form a sorted library, wherein the sorted librarycomprises candidate antibody variants with enhanced binding to thetarget molecule compared to the reference antibody; and (c) comparingthe frequency at which each amino acid residue alteration is present inthe display library and in the sorted library as determined by massivelyparallel sequencing, thereby determining whether each amino acid residuealteration is enriched in the sorted library compared to the displaylibrary, whereby the amino acid residue alteration is identified asconferring enhanced binding to the target molecule if it is enriched inthe sorted library compared to the display library.

In another aspect, the invention features a method of identifying anamino acid residue alteration that confers enhanced stability to anantibody, the method comprising: (a) providing a display librarycomprising nucleic acids encoding candidate antibody variants, whereineach candidate antibody variant comprises an amino acid residuealteration in the VH or the VL compared to a reference antibody, andwherein amino acid residue alterations at every position of the VH or VLare present in the display library; (b) sorting the display librarybased on binding of the candidate antibody variants to the targetmolecule to form a sorted library, wherein the sorted library comprisescandidate antibody variants with enhanced stability compared to thereference antibody; and (c) comparing the frequency at which each aminoacid residue alteration is present in the display library and in thesorted library as determined by massively parallel sequencing, therebydetermining whether each amino acid residue alteration is enriched inthe sorted library compared to the display library, whereby the aminoacid residue alteration is identified as conferring enhanced stabilityto the antibody if it is enriched in the sorted library compared to thedisplay library.

In some embodiments of any of the preceding aspects, the method furthercomprises determining the frequency at which each amino acid alterationis present in the display library and the sorted library by massivelyparallel sequencing following step (b).

In some embodiments of any of the preceding aspects, the amino acidresidue alteration is enriched at least 2-fold in the sorted librarycompared to the display library.

In some embodiments of any of the preceding aspects, the display libraryis selected from the group consisting of a phage display library, abacterial display library, a yeast display library, a mammalian displaylibrary, a ribosome display library, and an mRNA display library. Insome embodiments, the display library is a phage display library.

In some embodiments of any of the preceding aspects, the amino acidresidue alteration is encoded by a degenerate codon set. In someembodiments, the degenerate codon set is an NNK or an NNS codon set,wherein N is A, C, G, or T; K is G or T; and S is C or G. In someembodiments, the degenerate codon set is an NNK codon set.

In some embodiments of any of the preceding aspects, the sorting of step(b) comprises contacting the display library with an immobilized targetmolecule or epitope.

In some embodiments of any of the preceding aspects, the sorting of step(b) comprises contacting the display library with a soluble targetmolecule or epitope.

In some embodiments of any of the preceding aspects, the display librarycomprises at least 1×10⁶ candidate antibody variants. In someembodiments, the display library comprises at least 1×10⁸ antibodyvariants. In some embodiments, the display library comprises at least1×10⁹ antibody variants.

In some embodiments of any of the preceding aspects, the massivelyparallel sequencing comprises deep sequencing, ultra-deep sequencing,and/or next-generation sequencing.

In some embodiments of any of the preceding aspects, the antibody is amonoclonal antibody.

In some embodiments of any of the preceding aspects, the antibody is anIgG antibody.

In some embodiments of any of the preceding aspects, the antibody is anantibody fragment. In some embodiments, antibody fragment is selectedfrom the group consisting of Fab, scFv, Fv, Fab′, Fab′-SH, F(ab′)₂, anddiabody. In some embodiments, the antibody fragment is an Fab.

In some embodiments of any of the preceding aspects, the method furthercomprises generating an antibody that comprises an amino acid residuealteration identified by the steps of the method.

In some aspects, any one of the preceding antibodies can be used in themanufacture of a medicament for reducing or inhibiting angiogenesis in asubject having a disorder associated with pathological angiogenesis.

In some aspects, any one of the preceding antibodies can be used in themanufacture of a medicament for treating a disorder associated withpathological angiogenesis in a subject in need of such treatment.

In some aspects, any one of the preceding antibodies can be used in themanufacture of a medicament for inhibiting vascular permeability in asubject suffering from a disorder associated with undesirable vascularpermeability.

In some aspects, any one of the preceding antibodies can be used in amethod of reducing or inhibiting angiogenesis in a subject having adisorder associated with pathological angiogenesis.

In some aspects, any one of the preceding antibodies can be used in amethod of treating a disorder associated with pathological angiogenesisin a subject in need of such treatment.

In some aspects, any one of the preceding antibodies can be used in amethod of inhibiting vascular permeability in a subject suffering from adisorder associated with undesirable vascular permeability.

In another aspect, the invention features a composition comprising anyone of the preceding antibodies for use in a method of reducing orinhibiting angiogenesis in a subject having a disorder associated withpathological angiogenesis.

In another aspect, the invention features a composition comprising anyone of the preceding antibodies for use in a method of treating adisorder associated with pathological angiogenesis in a subject in needof such treatment.

In another aspect, the invention features a composition comprising anyone of the preceding antibodies for use in a method of inhibitingvascular permeability in a subject suffering from a disorder associatedwith undesirable vascular permeability.

In some aspects, any one of the preceding antibody conjugates can beused in the manufacture of a medicament for reducing or inhibitingangiogenesis in a subject having a disorder associated with pathologicalangiogenesis.

In some aspects, any one of the preceding antibody conjugates can beused in the manufacture of a medicament for treating a disorderassociated with pathological angiogenesis in a subject in need of suchtreatment.

In some aspects, any one of the preceding antibody conjugates can beused in a method of reducing or inhibiting angiogenesis in a subjecthaving a disorder associated with pathological angiogenesis.

In some aspects, any one of the preceding antibody conjugates can beused in a method of treating a disorder associated with pathologicalangiogenesis in a subject in need of such treatment.

In another aspect, the invention features a composition comprising anyone of the preceding antibody conjugates for use in a method of reducingor inhibiting angiogenesis in a subject having a disorder associatedwith pathological angiogenesis.

In another aspect, the invention features a composition comprising anyone of the preceding antibody conjugates for use in a method of treatinga disorder associated with pathological angiogenesis in a subject inneed of such treatment.

In some aspects, any one of the preceding fusion proteins can be used inthe manufacture of a medicament for reducing or inhibiting angiogenesisin a subject having a disorder associated with pathologicalangiogenesis.

In some aspects, any one of the preceding fusion proteins can be used inthe manufacture of a medicament for treating a disorder associated withpathological angiogenesis in a subject in need of such treatment.

In some aspects, any one of the preceding fusion proteins can be used ina method of reducing or inhibiting angiogenesis in a subject having adisorder associated with pathological angiogenesis.

In some aspects, any one of the preceding fusion proteins can be used ina method of treating a disorder associated with pathologicalangiogenesis in a subject in need of such treatment.

In another aspect, the invention features a composition comprising anyone of the preceding fusion proteins for use in a method of reducing orinhibiting angiogenesis in a subject having a disorder associated withpathological angiogenesis.

In another aspect, the invention features a composition comprising anyone of the preceding fusion proteins for use in a method of treating adisorder associated with pathological angiogenesis in a subject in needof such treatment.

It is to be understood that any of the embodiments described above withrespect to methods of treatment (e.g., with respect to antibodyproperties, additional therapeutic agents, disorders associated withpathological angiogenesis (e.g., ocular disorders such as AMD, DME, DR,or RVO), administration routes (e.g., intravitreal injection), and thelike) can be used in the context of the medicaments, uses, andcompositions described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1F are heatmaps showing the log 2 of the enrichment ratio (alsoreferred to as log 2 enrichment ratio) for all mutations in the panningsobtained from the NNK walk VH (FIGS. 1A-1C) and VL (FIGS. 1D-1F)libraries against anti-gD tag antibody (anti-gD), protein L, or proteinA. The amino acid sequence of wild-type G6.31 VH (FIGS. 1A-1C) or VL(FIGS. 1D-1F), beginning from position 2, is shown below each heat map.FIG. 1A shows the results obtained from panning the NNK walk VH libraryagainst anti-gD. FIG. 1B shows the results obtained from panning the NNKwalk VH library against protein L. FIG. 1C shows the results obtainedfrom panning the NNK walk VH library against protein A. FIG. 1D showsthe results obtained from panning the NNK walk VL library againstanti-gD. FIG. 1E shows the results obtained from panning the NNK walk VLlibrary against protein L. FIG. 1F shows the results obtained frompanning the NNK walk VL library against protein A.

FIGS. 2A-2B are a series of graphs and tables showing the correlationbetween log 2 enrichment ratios from panning of the VH (FIG. 2A) and theVL (FIG. 2B) against anti-gD-tag antibody (“gD”), protein A (“protA”),and protein L (“protL”). The tables show the Pearson correlationcoefficient (r²; “Cor”) for the comparison between (i) gD and protA;(ii) gD and protL; and (iii) protA and protL. The correlation forenrichment ratios of mutations in positions classified as belonging tothe hydrophobic core (“core”), extended hydrophobic core (“extendedcore”), VH/VL interface (“interface”), or positions which form importanthydrogen bonds, salt bridges, or are otherwise of interest (“other”) wasalso determined. These figures show that using an anti-gD antibody,protein A, or protein L to detect the folding of the Fab molecule onphage gives similar results. The only mutations which differedsignificantly in their enrichment ratios in the different pannings arethose located directly in the Protein A or Protein L binding sites.Those residues are labeled as “Protein A,” “Protein L 1,” and “Protein L2” (two binding sites for Protein L exist), respectively. The bindingsites for protein L are described in Graille et al. Structure 9(8):679-687, 2001, which is incorporated herein by reference in itsentirety. The binding sites for protein G are described in Graille etal. Proc. Natl. Acad. Sci. USA 97(10): 5399-5404, 2000, which isincorporated herein by reference in its entirety.

FIGS. 3A-3B are graphs showing the log 2 enrichment ratio for allmutations at a given position in the VH (FIG. 3A) and VL (FIG. 3B).Positions are colored according to whether they are located in thehydrophobic core (green), in the extended hydrophobic core (red), or inthe VH/VL interface (violet). Positions that are conserved and do nottolerate mutations (log 2 enrichment ratio Z-score <−0.5) are labeled.

FIGS. 3C-3D are renderings of the crystal structure of the antigen-freeG6 Fab (Protein Data Bank (PDB) code: 2FJF) showing the location ofconserved positions in the structure of the VH (FIG. 3C) and VL (FIG.3D), respectively. The color scheme is the same as in FIGS. 3A-3B. Inaddition, conserved positions which form important hydrogen bonds, saltbridges, or are otherwise important are colored pink.

FIGS. 4A-4B are heatmaps showing the log 2 enrichment ratio of allsingle amino acid substitutions obtained by panning the NNK walk VH(FIG. 4A) and VL (FIG. 4B) libraries against VEGF.

FIGS. 5A-5B are graphs showing that the log 2 enrichment ratios obtainedfrom VEGF panning of the VH (FIG. 5A) and VL (FIG. 5B) libraries have abi-modal distribution. Mutations which were depleted beyond thedetection limit were set to the maximum observed depletion for theparticular experiment. Mutations are colored accordingly to theirlocation in the HVRs (grey), in the conserved framework as identifiedusing the gD panning (orange), or the remainder of the framework (blue).

FIGS. 5C-5D are graphs showing a comparison between the log 2 enrichmentratio from VEGF panning of selected mutations with the change in Kdrelative to the parental antibody G6.31 (FIG. 5C) or the change inmelting temperature (T_(m)) relative to G6.31 (FIG. 5D). The same colorscheme as in FIGS. 5A-5B is used.

FIGS. 5E-5F are renderings of the crystal structure of the VEGF-boundform of the VH (FIG. 5E) and VL (FIG. 5F) of the G6 Fab (PDB code 2FJG)showing the location of selected mutations as spheres. The surface ofVEGF is shown as a surface representation. The same color scheme as inFIGS. 5A-5B is used. The label shows the change in T_(m) (° C.) and foldchange in binding affinity (Kd) compared to G6.31.

FIG. 6A is a visualization of superimposed renderings of moleculespresent in the crystal structure of the antigen-free G6 Fab (PDB 2FJF)showing the different Fab elbow angles exhibited by the molecules.Molecules exhibiting a small elbow angle (see, e.g., Example 3) arecolored in green, and molecules with a large elbow angle are colored inred.

FIGS. 6B-6C are renderings showing a detailed view of the VL-VHinterface and the side chain conformation of LC-83F and LC-1061 inmolecules with a small Fab elbow angle (FIG. 6B, green) and withmolecules with a large Fab elbow angle (FIG. 6C, red), respectively. Theβ-strand E, the helix α-1, and the loop connecting both elements areremoved for clarity.

FIG. 7A is a graph (right panel) showing the chi1 (_(X)1) angle ofposition LC-F83 from 319 human antibody structures from the PDB. Therendering (left panel) shows the position of LC-F83 in the “in” (lightred) and “out” (green) conformations.

FIG. 7B is a graph (right panel) showing the elbow angle backboneconformation for psi (4′) angle at position 105 and phi (ϕ) angle atposition 106. Structures are colored according to their chi1 angle asshown in FIG. 7A. The rendering (left panel) shows light chain (LC)positions 103-108 in the “in” (red) and “out” (green) conformations.

FIG. 7C is a series of graphs showing the elbow angle (top right) andVL/CL interface area (bottom right) for antibody structures with LC-F83in an “in”-conformation (red) and in an “out”-conformation (green). Theresults are compared with the Fab elbow angle and the VL/CL interfacesize of 319 human Fab structures from the PDB carrying LC-F83 and 22human structures carrying LC-83A. The left panel shows superimposedrenderings of G6 molecules having a large elbow angle (light red) and asmall elbow angle (green). The VL/CL interface area is indicated by thecircle.

FIG. 8A is a graph showing the chi1 angle for LC-F83 for a G6 Fabcrystal structure with a large elbow angle (G6 chains VU, G6-VU) and acrystal structure with a small elbow angle (G6 chains BA, G6-BA).

FIG. 8B is a graph showing the results of molecular dynamics simulationsfor the indicated molecules. The elbow angle is plotted as a function oftime.

FIG. 8C is a graph showing the results of molecular dynamics simulationsplotting Fab elbow angle as a function of VH/VL torsion angle(“HL-angle”). The scatter/contour plot shows the VH/VL torsion angle andelbow angle that VU.F83 (red) and VU.F83A (green) adopt during themolecular dynamics simulation. Two distinct populations are visible forthe two molecules.

FIG. 9A is a graph showing the Fab elbow angle distribution for themolecules BA-F83 (LC-F83 in an “out”-conformation), BA-F83A, VU-F93(LC-F83 in an “in”-conformation) and VU-F83A obtained during the last 75ns of a 100 ns molecular dynamics simulation. All samples weresignificantly different (p<0.001), except BA-F83 and BA-F83A, asdetermined by an analysis of variance (ANOVA)/Tukey's Honest significantdifference (HSD) test.

FIG. 9B is a graph showing the VH/VL torsion for five molecules ofG6_(unbound) with LC-F83 in an “out” conformation (red) and the fivemolecules with G6_(bound) with LC-F83 in an “in” conformation (green) aswell as the VH/VL torsion angle of the VEGF-bound G6 structures (“AGbound,” orange).

FIG. 9C is a graph showing the VH/VL torsion angle distribution of thesame molecules as in FIG. 9A during the same 100 ns molecular dynamicssimulation. All samples were significantly different (p<0.001), exceptBA-F83A and VU-F83A, as determined by an ANOVA/Tukey HSD test.

FIG. 9D is a rendering of the crystal structure of unbound G6 showingregions having different hydrogen-deuterium exchange patterns betweenG6.31 and G6.31_(LC-F83A). Regions colored in blue had slower exchangein G6.31_(LC-F83A) compared to G6.31, while regions colored in red hadfaster exchange in G6.31_(LC-F83A) compared to G6.31. The positions ofthe F83A mutation and the DE loop are indicated by lines.

FIG. 10A is a graph showing the distribution of somatic mutations forthe indicated VL positions of antibody sequences originating from IGKV1germlines. The mutational distribution in the upper panel was obtainedusing publicly available human antibody sequences from Genbank, theProtein Database (PDB), the Kabat database, the Abysis database, and theIMGT database (“Public dataset”). The mutational distribution in thelower panel was obtained using single molecule real-time sequencing(SMRT) of cDNA obtained from over 1000 individual human lymphoidtissues. The high mutation rates at the N-terminus of publicallyavailable sequences likely come from cloning artifacts.

FIG. 10B is a graph showing the most common mutations at position LC-83for IGKV1.39 sequences from the Public dataset described in FIG. 10A andExample 8 (Sanger) or the SMRT dataset (PacBio). IGKV1.39 carries aphenylalanine at position LC-83. Points are colored by the respectiveamino acid size. Large amino acids are colored in yellow and small aminoacids are colored in dark red.

FIG. 10C is a graph showing the most common mutations at position LC-106for all IGKJ sequences found in the Public dataset (Sanger) or in theSMRT dataset (PacBio). Points are colored by the respective amino acidsize. Large amino acids are colored in yellow and small amino acids arecolored in dark red.

FIG. 10D is a series of graphs showing affinity (Kd) of selected mutantvariants of G6.31 as measured by BIACORE® surface plasmon resonance(left panel) and the melting temperature T_(m) for selected G6.31 mutantvariants as determined by differential scanning fluorimetry (DSF) (rightpanel). The circles in the left panel represent the mean from threereplicates with the respective standard deviation shown by error bars.The circles in the right panel represent the mean from three replicatesand the respective standard deviation shown by error bars.

FIG. 11A is a graph showing the aqueous and vitreous pharmacokineticsfor each of G6.31 AAEE, G6.31 WT, and G6.31 AARR following intravitrealadministration of the respective Fab in rabbit eyes, as described inExample 11.

FIG. 11B is a graph showing the clearance from serum of each of G6.31AAEE, G6.31 WT, and G6.31 AARR following intravitreal administration ofthe respective Fab in rabbit eyes, as described in Example 11.

FIG. 12 is a table showing fragmentation analysis by capillaryelectrophoresis—sodium dodecyl sulfate (CE-SDS) for the indicatedantibody clones. Fragmentation is represented by the percentage of lowmolecular weight entities (% LMW) after 4 weeks, 12 weeks, and 24 weeksat 37° C. in PBS. The fragmentation is consistently reduced in all N94Avariants compared to wild-type G6.31. High molecular weight entities (%HMW) indicate impurities or aggregates. The main peak size in this assaydoes not correlate directly with the extent of fragmentation, butdepends on fragment size and extent of dye labeling. The anti-VEGF Fabranibizumab served as a control.

FIG. 13 is a graph showing a plot of inhibition of VEGF-induced HUVECmigration by the G6.31 variant LC-N94A compared to the parent G6.31 atvarying Fab concentrations, as described in Example 12.

FIG. 14 is a graph showing size exclusion chromatography (SEC) andrefractive index (RI) multi-angle light scattering (MALS) (SEC-RI-MALS)analysis of HA40K-rabFab, HA200K-rabFab, and HA600K-rabFab withweight-average molar mass (Mw) overlaid.

FIG. 15 is a graph showing that hyaluronic acid (HA) conjugated torabFab retains enzymatic susceptibility to digestion by hyaluronidase-2(HYAL2), as assessed by SEC-MALS analysis of HYAL-2-incubated HA andHA100K-rabFab. For HA-rabFab samples, the right Mw axis is expressed asMw of the HA component of the conjugate only.

FIG. 16 is a graph showing clearance of ranibizumab, rabFab, andHA100K-rabFab (“linear HA-rabFab”) from rabbit vitreous followingintravitreal injection. HA100K-rabFab displayed a half-life ofapproximately 11.9 days, compared to approximately 2.5 days for rabFab.

FIG. 17 is a graph showing that vitreal residence time is linearlycorrelated with hydrodynamic radius (Rh; indicated as “R_(H)” in thegraph) in rabbit vitreous. Historical data (filled circles) for rabFab,rabFab-20 kDa PEG, rabFab-40 kDa PEG and HA100K-rabFab can be used topredict half-lives for HA100K-G6.31 AARR (open circle), HA200K-G6.31AARR (open square) and HA300K-G6.31 (open triangle) based on measured Rhvalues.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs) and/or frameworkregions (FRs), compared to a parent antibody which does not possess suchalterations, such alterations resulting in an improvement in theaffinity of the antibody for antigen.

The term “vascular endothelial growth factor” or “VEGF” refers tovascular endothelial growth factor protein A, as exemplified by SEQ IDNO: 47 (see also Swiss Prot Accession Number P15692, Gene ID (NCBI):7422). The term “VEGF” encompasses the protein having the amino acidsequence of SEQ ID NO: 47 as well as homologues and isoforms thereof.The term “VEGF” also incompasses the known isoforms, e.g., spliceisoforms, of VEGF, e.g., VEGF₁₁₁, VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,and VEGF₂₀₆, together with the naturally-occurring allelic and processedforms thereof, including the 110-amino acid human vascular endothelialcell growth factor generated by plasmin cleavage of VEGF₁₆₅ as describedin Ferrara Mol. Biol. Cell. 21:687 (2010), Leung et al., Science,246:1306 (1989), and Houck et al., Mol. Endocrin., 5:1806 (1991). Theterm “VEGF” also refers to VEGFs from non-human species such as mouse,rat or primate. Sometimes the VEGF from a specific species are indicatedby terms such as hVEGF for human VEGF, mVEGF for murine VEGF, and thelike. The term “VEGF” is also used to refer to truncated forms of thepolypeptide comprising amino acids 8 to 109 or 1 to 109 of the 165-aminoacid human vascular endothelial cell growth factor. Reference to anysuch forms of VEGF may be identified in the present application, e.g.,by “VEGF₁₀₉,” “VEGF (8-109),” “VEGF (1-109)” or “VEGF₁₆₅.” The aminoacid positions for a “truncated” native VEGF are numbered as indicatedin the native VEGF sequence. For example, amino acid position 17(methionine) in truncated native VEGF is also position 17 (methionine)in native VEGF. The truncated native VEGF has binding affinity for theKDR and Flt-1 receptors comparable to native VEGF. The term “VEGFvariant” as used herein refers to a VEGF polypeptide which includes oneor more amino acid mutations in the native VEGF sequence. Optionally,the one or more amino acid mutations include amino acid substitution(s).For purposes of shorthand designation of VEGF variants described herein,it is noted that numbers refer to the amino acid residue position alongthe amino acid sequence of the putative native VEGF (provided in Leunget al., supra and Houck et al., supra). Unless specified otherwise, theterm “VEGF” as used herein indicates VEGF-A.

The terms “anti-VEGF antibody,” an “antibody that binds to VEGF,” and“antibody that specifically binds VEGF” refer to an antibody that iscapable of binding VEGF with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting VEGF. Inone embodiment, the extent of binding of an anti-VEGF antibody to anunrelated, non-VEGF protein is less than about 10% of the binding of theantibody to VEGF as measured, for example, by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to VEGF has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-VEGFantibody binds to an epitope of VEGF that is conserved among VEGF fromdifferent species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab-C, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments. In some instances, examples of antibody fragments include butare not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linearantibodies; single-chain antibody molecules (e.g., scFv); andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire light (L) chain along with the variableregion domain of the heavy (H) chain (VH), and the first constant domainof one heavy chain (CH1). Pepsin treatment of an antibody yields asingle large F(ab′)₂ fragment which roughly corresponds to two disulfidelinked Fab fragments having divalent antigen-binding activity and isstill capable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CH1 domain including one or more cysteines from the antibody hingeregion. Fab-C molecules are Fab molecules that are expressed such thatthe sequence is truncated at the first hinge cysteine, resulting in aFab with a free cysteine directly upon expression (see, e.g., Shatz etal. Mol. Pharmaceutics 2016; PubMed identifier (PMID) 27244474). Forexample, a Fab-C molecule may have a free cysteine at position Cys227 ofthe heavy chain. In other instances, a Fab-C molecule may have a freecysteine at position Cys229 of the heavy chain. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, noncovalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeHVRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor); and Bcell activation.

“Framework” or “framework region” or “FR” refers to variable domainresidues other than hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, FR residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies can comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. Thevariable or “V” domain mediates antigen binding and defines specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The term “hypervariable region” or“HVR” when used herein refers to the amino acid residues of an antibodywhich are responsible for antigen-binding. The hypervariable regiongenerally comprises amino acid residues from, for example, around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and aroundabout residues 26-35 (H1), 49-65 (H2) and 95-102 (H3) in the VH (in oneembodiment, H1 is around about residues 31-35); Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52(L2), and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2), and 96-101(H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). Thevariable domains of native heavy and light chains each comprise fourFRs, largely adopting a beta-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Accordingly, theHVR and FR sequences generally appear in the following sequence in VH(or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG₁ EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term an “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC). For a review of methods for assessment ofantibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007). In preferred embodiments, the antibody will bepurified (1) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (2) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes antibodies in situ within recombinant cells,because at least one component of the polypeptide natural environmentwill not be present. Ordinarily, however, isolated polypeptide will beprepared by at least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(VH) and a light chain variable domain (VL), where the VH-VL unit haspolyepitopic specificity (i.e., is capable of binding to two differentepitopes on one biological molecule or each epitope on a differentbiological molecule). Such multispecific antibodies include, but are notlimited to, full-length antibodies, antibodies having two or more VL andVH domains, antibody fragments such as Fab, Fab′. Fab-C. Fv, dsFv, scFv,diabodies, bispecific diabodies and triabodies, antibody fragments thathave been linked covalently or non-covalently. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s). “Dualspecificity” or “bispecificity” refers to the ability to specificallybind to two different epitopes on the same or different target(s).However, in contrast to bispecific antibodies, dual-specific antibodieshave two antigen-binding arms that are identical in amino acid sequenceand each Fab arm is capable of recognizing two antigens.Dual-specificity allows the antibodies to interact with high affinitywith two different antigens as a single Fab or IgG molecule. Accordingto one embodiment, the multispecific antibody in an IgG1 form binds toeach epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1μM to 0.001 pM, 0.5 μM to 0.001 pM or 0.1 μM to 0.001 pM. “Monospecific”refers to the ability to bind only one epitope.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

With regard to the binding of a antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a Kd for thetarget of 10⁻⁴ M or lower, alternatively 10⁻⁵ M or lower, alternatively10⁻⁶ M or lower, alternatively 10⁻⁷ M or lower, alternatively 10⁻⁸ M orlower, alternatively 10⁻⁹ M or lower, alternatively 10⁻¹⁰ M or lower,alternatively 10⁻¹¹ M or lower, alternatively 10⁻¹² M or lower or a Kdin the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁸ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹M. As will be appreciated by the skilled artisan, affinity and Kd valuesare inversely related. A high affinity for an antigen is measured by alow Kd value. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

A “nucleic acid encoding an anti-VEGF antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector.” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-VEGF antibody of the invention, an antibodyconjugate of the invention, a fusion protein of the invention, apolymeric formulation of the invention, or a nucleic acid encoding ananti-VEGF antibody of the invention) or a composition (e.g., apharmaceutical composition, e.g., a pharmaceutical composition includingan anti-VEGF antibody of the invention, an antibody conjugate of theinvention, a fusion protein of the invention, or a polymeric formulationof the invention) to a subject. The compositions utilized in the methodsdescribed herein can be administered, for example, intravitreally (e.g.,by intravitreal injection), by eye drop, intramuscularly, intravenously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularly, mucosally,intrapericardially, intraumbilically, intraocularly, intraorbitally,orally, topically, transdermally, by inhalation, by injection, byimplantation, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, by catheter, by lavage, incremes, or in lipid compositions. The compositions utilized in themethods described herein can also be administered systemically orlocally. The method of administration can vary depending on variousfactors (e.g., the compound or composition being administered and theseverity of the condition, disease, or disorder being treated).

“Angiogenesis” refers to the process through which new blood vesselsform from pre-existing blood vessels. Angiogenesis is distinct fromvasculogenesis, which is the de novo formation of endothelial cells frommesoderm cell precursors. Disorders associated with pathologicalangiogenesis can be treated by compositions and methods of theinvention. These disorders include both non-neoplastic disorders andcell proliferative disorders. Cell proliferative disorders include butare not limited those described below. Non-neoplastic disorders includebut are not limited to ocular conditions (non-limiting ocular conditionsinclude, for example, retinopathy including proliferative diabeticretinopathy, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema (DME), pathologic myopia, von Hippel-Lindaudisease, histoplasmosis of the eye, retinal vein occlusion (includingcentral (CRVO) and branched (BRVO) forms), corneal neovascularization,retinal neovascularization, retinopathy of prematurity (ROP), familialexudative vitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, andhypertensive retinopathy), autoimmune diseases (e.g., rheumatoidarthritis (RA), psoriasis, ankylosing spondylitis, and inflammatorybowel disease (e.g., Crohn's disease and ulcerative colitis)), undesiredor aberrant hypertrophy, arthritis, psoriatic arthritis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, arterialarteriosclerosis, vascular restenosis, arteriovenous malformations(AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,lung inflammation, acute lung injury/ARDS, sepsis, primary pulmonaryhypertension, malignant pulmonary effusions, cerebral edema (e.g.,associated with acute stroke/closed head injury/trauma), synovialinflammation, pannus formation in RA, myositis ossificans, hypertropicbone formation, osteoarthritis (OA), refractory ascites, polycysticovarian disease, endometriosis, 3rd spacing of fluid diseases(pancreatitis, compartment syndrome, burns, bowel disease), chronicasthma, uterine fibroids, premature labor, chronic inflammation such asIBD (Crohn's disease and ulcerative colitis), inflammatory renaldiseases (including glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy andhypertensive nephrosclerosis), diseases occurring after transplants,renal allograft rejection, inflammatory diseases, nephrotic syndrome,undesired or aberrant tissue mass growth (non-cancer), hemophilicjoints, hypertrophic scars, inhibition of hair growth, Osler-Webersyndrome, pyogenic granuloma retrolental fibroplasias, scleroderma,trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia,ascites, pericardial effusion (such as that associated withpericarditis), and pleural effusion. Additional ocular disorders aredescribed below.

Other disorders which may be associated with pathological angiogenesisinclude nonunion fractures (fractures that will not heal), pyogenicgranuloma, trachoma, hemophilic joints, vascular adhesions andhypertrophic scars, graft rejection, fibrovascular tissue, acne rosacea,acquired immune deficiency syndrome, artery occlusion, atopic keratitis,bacterial ulcers, Bechet's disease, carotid obstructive disease, chronicinflammation, chronic retinal detachment, chronic uveitis, chronicvitritis, contact lens overwear, corneal graft rejection, corneal graftneovascularization, Eales disease, epidemic keratoconjunctivitis, fungalulcers, Herpes simplex infections, Herpes zoster infections,hyperviscosity syndromes, Kaposi's sarcoma, leukemia, lipiddegeneration, Lyme's disease, marginal keratolysis, Mooren ulcer,Mycobacteria infections other than leprosy, myopia, optic pits,osteoarthritis, Paget's disease, pars planitis, pemphigoid,phylectenulosis, polyarteritis, post-laser complications, protozoaninfections, pseudoxanthoma elasticum, pterygium keratitis sicca, radialkeratotomy, retrolental fibroplasias, sarcoid, scleritis, sickle cellanemia, Sjogren's syndrome, Stargarts disease, Steven's Johnson disease,superior limbic keratitis, syphilis, systemic lupus, Temren's marginaldegeneration, toxoplasmosis, trauma, vein occlusion, Vitamin Adeficiency and Wegeners sarcoidosis, undesired angiogenesis associatedwith diabetes, parasitic diseases, abnormal wound healing, hypertrophyfollowing surgery, injury or trauma, inhibition of hair growth,inhibition of ovulation and corpus luteum formation, inhibition ofimplantation and inhibition of embryo development in the uterus.

The term “ocular disorder,” as used herein, includes any ocular disorder(also referred to interchangeably herein as “ocular condition”)associated with pathogical angiogenesis. An ocular disorder may becharacterized by altered or unregulated proliferation and/or invasion ofnew blood vessels into the structures of ocular tissues such as theretina or cornea. Non-limiting ocular disorders include, for example,AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, andgeographic atrophy (GA)), macular degeneration, macular edema, DME(e.g., focal, non-center DME and diffuse, center-involved DME),retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g, allergic) conjunctivitis), Leber congenitalamaurosis (also known as Leber's congenital amaurosis or LCA), uveitis(including infectious and non-infectious uveitis), choroiditis (e.g.,multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye,traumatic eye injury, Sjögren's disease, and other ophthalmic diseaseswherein the disease or disorder is associated with ocularneovascularization, vascular leakage, and/or retinal edema. Additionalexemplary ocular disorders include diseases associated with rubeosis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue, including all forms ofproliferative vitreoretinopathy.

Exemplary diseases associated with corneal neovascularization include,but are not limited to, epidemic keratoconjunctivitis, vitamin Adeficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, terygium keratitis sicca, Sjögren's syndrome, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, marginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome,periphigoid radial keratotomy, and corneal graph rejection.

Exemplary diseases associated with retinal/choroidal neovascularizationinclude, but are not limited to, diabetic retinopathy, maculardegeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum, Paget's disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infections,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, retinitis pigmentosa, retina edema (including macularedema), Eales disease, Behcet's disease, infections causing retinitis orchoroiditis (e.g., multifocal choroidits), presumed ocularhistoplasmosis, Best's disease (vitelliform macular degeneration),myopia, optic pits, Stargart's disease, pars planitis, retinaldetachment (e.g., chronic retinal detachment), hyperviscosity syndromes,toxoplasmosis, trauma, and post-laser complications.

“Disorders associated with undesirable vascular permeability,” as usedherein, include, for example, edema associated with brain tumors,ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion, pleuraleffusion, permeability associated with cardiovascular diseases such asthe condition following myocardial infarctions and strokes and the like.

It is to be understood that the classifications described above are notmutually exclusive, and a disorder may fall under multiple categories.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

The terms “cancer”, “cancerous”, “cell proliferative disorder”,“proliferative disorder” and “tumor” are not mutually exclusive asreferred to herein.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stabiliy of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, PIGF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Del-1,fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,Placental growth factor, Platelet-derived endothelial cell growth factor(PD-ECGF), Platelet-derived growth factor, especially PDGF-BB orPDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin, Transforminggrowth factor-alpha (TGF-alpha), Transforming growth factor-beta(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascularendothelial growth factor (VEGF)/vascular permeability factor (VPF),etc. It would also include factors that accelerate wound healing, suchas growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermalgrowth factor (EGF), CTGF and members of its family, and TGF-alpha andTGF-beta. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol.,53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003);Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini etal., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing knownangiogenic factors); and Sato, Int. J. Clin. Oncol., 8:200-206 (2003).

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, an polynucleotide, an polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as defined above,e.g., VEGF antagonists (e.g., antibodies to VEGF-A or to the VEGF-Areceptor (e.g., KDR receptor or Flt-1 receptor)), PDGF antagonists(e.g., anti-PDGFR inhibitors such as GLEEVEC™ (Imatinib Mesylate)).Anti-angiogenesis agents also include native angiogenesis inhibitors,e.g., angiostatin, endostatin, etc. See, for example, Klagsbrun andD'Amore. Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar.Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)(e.g., Table 2 listing known antiangiogenic factors): and, Sato Int. J.Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists anti-angiogenicagents used in clinical trials).

The term “VEGF antagonist,” as used herein, refers to a molecule capableof binding to VEGF, reducing VEGF expression levels, or neutralizing,blocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities, including, but not limited to, VEGF binding toone or more VEGF receptors, VEGF signaling, and VEGF-mediatedangiogenesis and endothelial cell survival or proliferation. Forexample, a molecule capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities canexert its effects by binding to one or more VEGF receptor (VEGFR) (e.g.,VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR)). Included as VEGF antagonists useful inthe methods of the invention are polypeptides that specifically bind toVEGF, anti-VEGF antibodies and antigen-binding fragments thereof,receptor molecules and derivatives which bind specifically to VEGFthereby sequestering its binding to one or more receptors, fusionsproteins (e.g., VEGF-Trap (Regeneron)), and VEGF₁₂₁-gelonin (Peregrine).VEGF antagonists also include antagonist variants of VEGF polypeptides,antisense nucleobase oligomers complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide; small RNAscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; ribozymes that target VEGF; peptibodies to VEGF; andVEGF aptamers. VEGF antagonists also include polypeptides that bind toVEGFR, anti-VEGFR antibodies, and antigen-binding fragments thereof, andderivatives which bind to VEGFR thereby blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities(e.g., VEGF signaling), or fusions proteins. VEGF antagonists alsoinclude nonpeptide small molecules that bind to VEGF or VEGFR and arecapable of blocking, inhibiting, abrogating, reducing, or interferingwith VEGF biological activities. Thus, the term “VEGF activities”specifically includes VEGF-mediated biological activities of VEGF. Incertain embodiments, the VEGF antagonist reduces or inhibits, by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, theexpression level or biological activity of VEGF. In some embodiments,the VEGF inhibited by the VEGF-specific antagonist is VEGF (8-109), VEGF(1-109), or VEGF₁₆₅.

As used herein VEGF antagonists can include, but are not limited to,anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab,tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules(e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), andziv-aflibercept (VEGF Trap; ZALTRAP®)), bispecific VEGF antibodies(e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodiesdisclosed in US 2001/0236388), bispecific antibodies includingcombinations of two of anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms,anti-VEGF antibodies (e.g., bevacizumab, sevacizumab, and ranibizumab),and nonpeptide small molecule VEGF antagonists (e.g., pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, and tivozanib). Additional VEGFantagonists are described below.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human. A “subject” may be a “patient.”

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals,nonhuman primates, and zoo, sports, or pet animals, such as dogs,horses, cats, cows, etc.

A “disorder” is any condition that would benefit from treatment with theantibody. For example, mammals who suffer from or need prophylaxisagainst abnormal angiogenesis (excessive, inappropriate or uncontrolledangiogenesis) or vascular permeability. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose the mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include disorders associated withpathological angiogenesis (e.g., ocular disorders and cell proliferativedisorders) and disorders associated with undesirable vascularpermeability.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib(TARCEVA™), platelet derived growth factor inhibitors (e.g., GLEEVEC™(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons,cytokines, antagonists (e.g., neutralizing antibodies) that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS,APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive andorganic chemical agents, and the like. Combinations thereof are alsoincluded in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm⁵³,Bi²¹², P32, Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includechemical compounds useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1l and calicheamicinomegal1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paditaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg. Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)(including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylomithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (TARCEVA™)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rlL-2; LURTOTECAN® topoisomerase 1 inhibitor,ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids, or derivativesof any of the above.

The term “prodrug” as used herein refers to a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient (e.g., an anti-VEGF antibody, an antibody conjugate, a fusionprotein, or a polymeric formulation) contained therein to be effective,and which contains no additional components which are unacceptably toxicto a subject to which the formulation would be administered.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention or other compositions that include an antibody of theinvention (e.g., an antibody conjugate, a fusion protein, or a polymericformulation) are used to delay development of a disease or to slow theprogression of a disease.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the antibody where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (e.g., anti-VEGF antibodies of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

A “mutation” is a deletion, insertion, or substitution of anucleotide(s) relative to a reference nucleotide sequence, such as awild-type sequence.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,eg. NNK, NNS, XYZ, DVK and the like. Synthesis of oligonucleotides withselected nucleotide “degeneracy” at certain positions is well known inthat art, for example the TRIM approach (Knappek et al., J. Mol. Biol.296:57-86 (1999)); Garrard et al., Gene 128:103 (1993)). Such sets ofoligonucleotides having certain codon sets can be synthesized usingcommercial nucleic acid synthesizers (available from, for example,Applied Biosystems, Foster City, Calif.), or can be obtainedcommercially (for example, from Life Technologies, Rockville, Md.).Therefore, a set of oligonucleotides synthesized having a particularcodon set will typically include a plurality of oligonucleotides withdifferent sequences, the differences established by the codon set withinthe overall sequence. Oligonucleotides, as used according to theinvention, have sequences that allow for hybridization to a variabledomain nucleic acid template and also can, but does not necessarily,include restriction enzyme sites useful for, for example, cloningpurposes.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, for example, filamentous phage, particles. Autility of phage display lies in the fact that large libraries ofrandomized protein variants can be rapidly and efficiently sorted forthose sequences that bind to a target antigen with high affinity.Display of peptide and protein libraries on phage has been used forscreening millions of polypeptides for ones with specific bindingproperties. Polyvalent phage display methods have been used fordisplaying small random peptides and small proteins through fusions toeither gene III or gene VIII of filamentous phage. Wells and Lowman,Curr. Opin. Struct. Biol., 3:355-362 (1992), and references citedtherein. In a monovalent phage display, a protein or peptide library isfused to a gene III or a portion thereof, and expressed at low levels inthe presence of wild-type gene III protein so that phage particlesdisplay one copy or none of the fusion proteins. Avidity effects arereduced relative to polyvalent phage so that sorting is on the basis ofintrinsic ligand affinity, and phagemid vectors are used, which simplifyDNA manipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 3:205-0216 (1991).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, for example, Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

A “variant” or “mutant” of a starting or reference polypeptide (e.g., areference antibody or its variable domain(s)/HVR(s)), is a polypeptidethat (1) has an amino acid sequence different from that of the startingor reference polypeptide and (2) was derived from the starting orreference polypeptide through either natural or artificial (man-made)mutagenesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequence of the polypeptide of interest, referred to herein as “aminoacid residue alterations.” Thus, a variant HVR refers to a HVRcomprising a variant sequence with respect to a starting or referencepolypeptide sequence (such as that of a source antibody or antigenbinding fragment). An amino acid residue alteration, in this context,refers to an amino acid different from the amino acid at thecorresponding position in a starting or reference polypeptide sequence(such as that of a reference antibody or fragment thereof). Anycombination of deletion, insertion, and substitution may be made toarrive at the final variant or mutant construct, provided that the finalconstruct possesses the desired functional characteristics. The aminoacid changes also may alter post-translational processes of thepolypeptide, such as changing the number or position of glycosylationsites.

A “wild-type (WT)” or “reference” sequence or the sequence of a“wild-type” or “reference” protein/polypeptide, such as an HVR or avariable domain of a reference antibody, may be the reference sequencefrom which variant polypeptides are derived through the introduction ofmutations. In general, the “wild-type” sequence for a given protein isthe sequence that is most common in nature. Similarly, a “wild-type”gene sequence is the sequence for that gene which is most commonly foundin nature. Mutations may be introduced into a “wild-type” gene (and thusthe protein it encodes) either through natural processes or throughman-induced means. The products of such processes are “variant” or“mutant” forms of the original “wild-type” protein or gene.

A “reference antibody,” as used herein, refers to an antibody orfragment thereof whose antigen-binding sequence serves as the templatesequence upon which diversification according to the criteria describedherein is performed. An antigen-binding sequence generally includes anantibody variable region, preferably at least one HVR, preferablyincluding framework regions.

By “massively parallel sequencing” or “massive parallel sequencing,”also known in the art as “next-generation sequencing,” or “secondgeneration sequencing,” is meant any high-throughput nucleic acidsequencing approach. These approaches typically involve parallelsequencing of a large number (e.g., thousands, millions, or billions) ofspatially separated, clonally amplified DNA templates or single DNAmolecules. See, for example, Metzker, Nature Reviews Genetics 11: 31-36,2010.

“Enriched,” as used herein, means that an entity (e.g., an amino acidresidue alteration) is present at a higher frequency in a sorted libraryas compared to a corresponding reference library (e.g., an unsortedlibrary, or a library that has been sorted for a different ornon-relevant antigen). In contrast, “depleted” means that an entity (forexample, an amino acid residue alteration) is present at a lowerfrequency in a sorted library as compared to a corresponding referencelibrary (e.g., an unsorted library, or a library that has been sortedfor a different or non-relevant antigen). The term “neutral,” when usedin reference to methods of identifying amino acid residue variants,means that an entity is neither enriched nor depleted, in other words,it is present at approximately the same frequency in a sorted library ascompared to a corresponding reference library (e.g., an unsortedlibrary, or a library that has been sorted for a different ornon-relevant antigen).

By “isoelectric point (pI)” is meant the pH at which a molecule (e.g., aprotein, such as an antibody) carries no net electrical charge, alsoreferred to in the art as “pH(I)” or “IEP.”

As used herein, an “antibody conjugate” is an antibody covalentlyattached to one or more polymers. Any suitable polymer may be conjugatedto an antibody, for example, a hydrophilic polymer (e.g., hyaluronicacid (HA) or polyethylene glycol (PEG)) or a hydrophobic polymer (e.g.,poly(lactic-co-glycolic acid) (PLGA)).

As used herein, the term “polymer” means a molecule that includesrepeating structural units (i.e., monomers) connected by chemical bondsin a linear, circular, branched, crosslinked, or dendrimeric manner, ora combination thereof. A polymer may be synthetic or naturallyoccurring, or a combination thereof. It is to be understood that theterm “polymer” encompasses copolymers, which are polymers that includetwo or more different monomers. A polymer may also be a homopolymer,which is a polymer that includes only a single type of monomer.

The terms “hyaluronic acid,” “hyaluranon,” and “HA.” which are usedinterchangeably herein, refer to a polymeric glycosaminoglycan (GAG),which contains repeating disaccharide units of N-acetyl glucosamine andglucuronic acid. HA is an anionic, nonsulfated GAG, which can be found,for example, in extracellular matrix (e.g., in the vitreous of the eye),connective tissue, epithelial, and neural tissue.

The term “polyethylene glycol” or “PEG” as used herein, refers to apolyether compound that is also known as polyethylene oxide (PEO) orpolyoxyethylene (POE), depending on its molecular weight. PEG may have astructure of H—(O—CH₂—CH₂)_(n)—OH, wherein n is any suitable integer.The PEG may be a branched PEG, a star PEG, or a comb PEG. The PEG maybe, for example, a PEG tetramer, a PEG hexamer, or a PEG octamer.

As used herein, the term “fusion protein” refers to a protein in which afirst peptide, protein, or polypeptide, e.g., an antibody (e.g., ananti-VEGF antibody (e.g., any anti-VEGF antibody described herein, e.g.,G6.31 AARR)) is linked, directly or indirectly, to a second peptide,protein, or polypeptide, e.g., an ocular binding domain (e.g., an HAbinding domain). In one example, the first peptide, protein, orpolypeptide (e.g., an antibody) may be linked to the second peptide,protein, or polypeptide (e.g., an ocular binding domain (e.g., an HAbinding domain)) by a linker. In the context of fusion proteins, theterms “links” and “linked,” and grammatical variations thereof, are usedinterchangeably with the term “covalently attached,” and refer to adirect or indirect covalent bonding (e.g., a peptide bond) between twomoieties of the fusion protein. In general, the fusion proteins of theinvention are antibody fusion proteins. The antibody may be an antibodyfragment, for example, an Fab, an Fab′, or an Fab-C. In some instances,the antibody fragment is an Fab.

The term “ocular binding domain” refers to a peptide, protein,polypeptide or fragment thereof that binds to a biological substancefound in the eye (e.g., the cornea, vitreous, retina, retina pigmentepithelium, or choroid). As one example, in some instances, thebiological substance found in the eye is an extracellular matrixcomponent, for example, a carbohydrate (e.g., a charged carbohydrate(e.g., a glycosaminoglycan)), a glycoprotein (e.g., fibrillin andopticin), or a protein (e.g., a collagen (e.g., collagen types I-XXVII,particularly collagen II, collagen IX, collagen V, collagen VI, collagenXI, and heterotypic collagen fibrils thereof), or other extracellularmatrix components described, for example, in Le Goff et al., Eye22:1214-1222, 2008. In some instances, the extracellular matrixcomponent is a glycosaminoglycan, for example, HA or a proteoglycan(e.g., chondroitin sulfate or heparin sulfate). In one example, theocular binding domain is a hyaluronic acid binding domain.

As used herein, the term “hyaluronic acid binding domain” or “HA bindingdomain” refers to a peptide, protein, polypeptide, or fragment thereofthat binds HA. An HA binding domain may be derived from an HA bindingprotein (also referred to in the art as a “hyaladherin”), including, forexample, tumor necrosis factor-stimulated gene 6 (TSG6), lymphaticvessel endothelial hyaluronan receptor 1 (LYVE-1), hyaluronan andproteoglycan link protein (HAPLN) 1, HAPLN2, HAPLN3, HAPLN4, aggrecan,brevican, neurocan, phosphacan, versican, CAB61358, KIA0527, stabilin-1,stabilin-2, RHAMM, bacterial HA synthase, and collagen VI. Other HAbinding proteins are known in the art. Exemplary HA binding domainsinclude link modules, G1 domains, and lysine-rich oligopeptides.

A “link module” (also referred to in the art as a “link domain”) is astructural domain of approximately 100 amino acids (see, e.g., Yang etal. EMBO J., 13(2): 286-296; Mahoney et al., J. Biol. Chem. 276(25):22764-22771, 2001; and Blundell et al. J. Biol. Chem. 278(49):49261-49270, 2003) that binds HA. Exemplary, non-limiting link modulesinclude those from TSG6, CD44, LYVE-1, HAPLN1, HAPLN2, HAPLN3, HAPLN4,aggrecan, brevican, neurocan, phosphacan, versican, CAB61358, KIA0527,stabilin-1, and stabilin-2 link modules, or variants thereof. As oneexample, the link module of the HA binding protein TSG6 may includeamino acid residues 36-128 of human TSG6 (UniProt Accession No. P98066).A variant link module binds HA and may have, for example, at least 80%amino acid sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher sequence identity) to a wild-type orreference link module, and may include sequence variations such asinsertions, deletions, and substitutions (e.g., conservative amino acidsubstitutions) relative to the wild-type or reference link module aminoacid sequence.

In the context of fusion proteins, a “linker” may be a peptide orpolypeptide that links (e.g., covalently links) a first moiety (e.g., anantibody (e.g., an anti-VEGF antibody (e.g., any anti-VEGF antibodydescribed herein, e.g., G6.31 AARR))) to a second moiety (e.g., anocular binding domain (e.g., an HA binding domain)). A linker mayinclude an amino acid sequence of any suitable length, for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore amino acid residues. In some instances, a linker includes about 3,about 4, about 5, about 6, about 7, or about 8 residues. In someinstances, a linker includes the amino acid sequence of GGGGS (SEQ IDNO: 61).

A “hydrogel,” as used herein, refers to a hydrophilic or amphiphilicpolymeric network composed of homopolymers or copolymers, which isinsoluble due to the presence of covalent chemical crosslinks. Thecrosslinks may provide the network structure and physical integrity.Hydrogels may exhibit a thermodynamic compatibility with water, whichallows them to swell in aqueous media.

As used herein, the term “reversible prodrug linker” means a moiety thatis attached on one end to a biologically active moiety (e.g., a drug,such as an anti-VEGF antibody), through a reversible linkage, and isattached on another end through a permanent bond to a carrier (e.g., ahydrogel), thereby linking the biologically active moiety to thecarrier. Such reversible prodrug linkers are non-enzymaticallyhydrolytically degradable, i.e., cleavable, under physiologicalconditions (e.g., aqueous buffer at pH 7.4, 37° C.) with half-livesranging from, for example, one hour to twelve months. Reversiblelinkages include, for example, aconityls, acetals, amides, carboxylicanhydrides, esters, imines, hydrazones, maleamic acid amides, orthoesters, phosphamides, phosphoesters, phosphosilyl esters, silyl esters,sulfonic esters, aromatic carbamates, and combinations thereof. Incontrast, permanent linkages are non-enzymatically hydrolyticallydegradable under physiological conditions (aqueous buffer at pH 7.4, 37°C.) with half-lives of more than twelve months. Exemplary reversibleprodrug linkers are described, for example, in International PatentApplication Publication No. WO 2014/056923, which is incorporated hereinby reference in its entirety.

The term “clearance,” as used herein, refers to the volume of asubstance (e.g., an anti-VEGF antibody, an antibody conjugate, a fusionprotein (e.g., a Fab fusion protein), or a polymeric formulation)cleared from a compartment (e.g., the eye (e.g., the vitreous)) per unittime.

The term “half-life” refers to the time required for the concentrationof a substance (e.g., an anti-VEGF antibody, an antibody conjugate, afusion protein (e.g., a Fab fusion protein), or a polymeric formulation)to decrease by one-half, in vivo (e.g., in the eye (e.g., the vitreous))or in vitro.

II. Compositions and Methods

The invention provides novel antibodies that bind to VEGF, and methodsof making and using the same, for example, for diagnostic andtherapeutic uses. The invention also provides compositions that includeanti-VEGF antibodies (including any anti-VEGF antibody describedherein), including antibody conjugates, fusion proteins, and polymericformulations, as well as methods of making and using the same, forexample, for diagnostic and therapeutic uses. The invention alsoprovides methods of identifying antibody variants with improvedproperties, for example, enhanced binding affinity, stability, and/orexpression.

A. Exemplary Anti-VEGF Antibodies

In one aspect, the invention is based, in part, on antibodies thatspecifically bind to VEGF. Antibodies of the invention are useful, forexample, for reducing angiogenesis and for treating or delaying theprogression of a disorder associated with pathological angiogenesis(e.g., ocular disorders or cell proliferative disorders). Antibodies ofthe invention are also useful, for example, for inhibiting vascularpermeability and treating disorders associated with undesirable vascularpermeability.

In some instances, the anti-VEGF antibody may include at least one, two,three, four, five, or six HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2comprising the amino acid sequence of GX₁TPX₂GGX₃X₄X₅YX₆DSVX₇X₈ (SEQ IDNO: 2), wherein X₁ is lie or His, X₂ is Ala or Arg, X₃ is Tyr or Lys, X₄is Thr or Glu, X₅ is Arg, Tyr, Gln, or Glu, X₆ is Ala or Glu, X₇ is Lysor Glu, and X₈ is Gly or Glu; (c) an HVR-H3 comprising the amino acidsequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising theamino acid sequence of RASQX₁VSTAVA (SEQ ID NO: 4), wherein X₁ is Asp orArg; (e) an HVR-L2 comprising the amino acid sequence of X₁ASFLYS (SEQID NO: 5), wherein X₁ is Ser or Met; and (f) an HVR-L3 comprising theamino acid sequence of X₁QGYGX₂PFT (SEQ ID NO: 6), wherein X₁ is Gln,Asn, or Thr and X₂ is Ala, Asn, Gln, or Arg, or a combination of one ormore of the above HVRs and one or more variants thereof having at leastabout 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity)to any one of SEQ ID NOs: 1-6.

For instance, the anti-VEGF antibody may include at least one, two,three, four, five, or six HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7),GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG (SEQ ID NO: 22);(c) an HVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ IDNO: 3); (d) an HVR-L1 comprising the amino acid sequence of RASQDVSTAVA(SEQ ID NO: 8); (e) an HVR-L2 comprising the amino acid sequence ofSASFLYS (SEQ ID NO: 9): and (f) an HVR-L3 comprising the amino acidsequence of QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 7-10, or21-23.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQID NO: 7); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, or 7-10. In aparticular example, in some instances, the anti-VEGF antibody includesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acidsequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprisingthe amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) anHVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and(f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ IDNO: 10).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (t) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 11 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYEYYADSVEG (SEQID NO: 22); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 8, 9, 22, or23. In a particular example, in some instances, the anti-VEGF antibodyincludes the following six HVRs: (a) an HVR-H1 comprising the amino acidsequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the aminoacid sequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) anHVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8);(e) an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO:9); and (f) an HVR-L3 comprising the amino acid sequence of QQGYGNPFT(SEQ ID NO: 23).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 24); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (t) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 38.

In some instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 38.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYEYYADSVEG (SEQID NO: 22); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 8-10, or 22.In a particular example, in some instances, the anti-VEGF antibodyincludes the following six HVRs: (a) an HVR-H1 comprising the amino acidsequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the aminoacid sequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) anHVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8);(e) an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO:9); and (f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT(SEQ ID NO: 10).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 34.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 35.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (t) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 35.

For example, in yet other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 36.

For example, in still further instances, the anti-VEGF antibody includesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acidsequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprisingthe amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) anHVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and(f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ IDNO: 10). In some instances, the anti-VEGF antibody includes thefollowing four heavy chain variable domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO:29); (b) an FR-H2 comprising the amino acid sequence of WVRQEPGEGLEWVA(SEQ ID NO: 30); (c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 37.

In other instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 37.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

In other instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 51); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

In some instances, the anti-VEGF antibody comprises (a) a heavy chainvariable (VH) domain comprising an amino acid sequence having at least90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity) to, or the sequence of, any one of SEQ IDNOs: 11, 40, or 42; (b) a light chain variable (VL) domain comprising anamino acid sequence having at least 90% sequence (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, any one of SEQ ID NOs: 12, 41, or 46; or (c) a VH domain asin (a) and a VL domain as in (b). For example, in some instances, theantibody comprises a VH domain comprising the amino acid sequence of SEQID NO: 11 and a VL domain comprising the amino acid sequence of SEQ IDNO: 12. In some instances, the antibody comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence of SEQ ID NO: 12. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:42 and a VL domain comprising the amino acid sequence of SEQ ID NO: 12.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence of SEQ ID NO: 41. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:11 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46.

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14) orWVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQID NO: 45); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), orGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20) orFGQGTKVEVK (SEQ ID NO: 55).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60). For example, insome instances, the anti-VEGF antibody comprises (a) a VH domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 11; (b) a VL domaincomprising an amino acid sequence having at least 90% sequence (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, SEQ ID NO: 11; or (c) a VH domain as in (a) anda VL domain as in (b). In some instances, the anti-VEGF antibody mayinclude (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQID NO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following heavychain framework regions: (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16). Insome instances, the anti-VEGF antibody includes the following lightchain framework regions: (a) an FR-L1 comprising the amino acid sequenceof DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20). In some instances, the anti-VEGF antibodyincludes a binding domain comprising (a) a VH domain comprising an aminoacid sequence of SEQ ID NO: 11 and (b) a VL domain comprising an aminoacid sequence of SEQ ID NO: 12. In some instances, the exemplaryanti-VEGF is N94A.F83A.N82aR.Y58R.

In some instances, the anti-VEGF antibody comprises (a) VH domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 33 or 51; (b) a VL domaincomprising an amino acid sequence having at least 90% sequence (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, any one of SEQ ID NOs: 12, 34, 35, 36, 37, or38; or (c) a VH domain as in (a) and a VL domain as in (b). For example,in some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 12. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 35. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of SEQ ID NO: 38. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:51 and a VL domain comprising the amino acid sequence of SEQ ID NO: 35.n some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:51 and a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): an FR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30) orWVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) anFR-H4 comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 48 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.In certain embodiments, the antibody is G6.31 AARR expressed in Fabformat.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 49 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.In certain embodiments, the antibody is a variant version of G6.31 AARRthat lacks reactivity to anti-human IgG.

In a further aspect, an anti-VEGF antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-8 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g.,from 10⁻⁹ M to 10⁻¹³ M). For example, in some instances, an antibodyprovided herein binds human VEGF (hVEGF) with a Kd of about 10 nM orlower. In some instances, an antibody provided herein binds hVEGF with aKd of about 5 nM or lower. In some instances, an antibody providedherein binds hVEGF with a Kd of about 2 nM or lower. For example, insome instances, the antibody binds hVEGF with a Kd between about 25 pMand about 2 nM (e.g., about 25 pM, about 50 pM, about 75 pM, about 100pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225pM, about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350pM, about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475pM, about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600pM, about 625 pM, about 650 pM, about 675 pM, about 700 pM, about 725pM, about 750 pM, about 775 pM, about 800 pM, about 825 pM, about 850pM, about 875 pM, about 900 pM, about 925 pM, about 950 pM, about 975pM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM,about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, orabout 2 nM). In some instances, the antibody binds hVEGF with a Kdbetween about 75 pM and about 600 pM (e.g., about 75 pM, about 100 pM,about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM,about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM,about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM,about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM).In some instances, the antibody binds hVEGF with a Kd between about 75pM and about 500 pM. In some instances, the antibody binds hVEGF with aKd between about 75 pM and about 400 pM. In some instances, the antibodybinds hVEGF with a Kd between about 75 pM and about 300 pM. In someinstances, the antibody binds hVEGF with a Kd between about 75 pM andabout 200 pM. In some instances, the antibody binds hVEGF with a Kdbetween about 75 pM and about 150 pM. In some instances, the antibodybinds hVEGF with a Kd between about 75 pM and about 125 pM. In someinstances, the antibody binds hVEGF with a Kd between about 75 pM andabout 100 pM. In some instances, the antibody binds hVEGF with a Kd ofabout 80 pM. In some instances, the antibody binds hVEGF with a Kd ofabout 60 pM. In some instances, the antibody binds hVEGF with a Kd ofabout 40 pM.

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see. e.g., Chen et al., J. Mol.Biol. 293:865-881 (1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin (BSA) in phosphate buffered saline (PBS) for two to fivehours at room temperature (approximately 23° C.). In a non-adsorbentplate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed withserial dilutions of a Fab of interest (e.g., consistent with assessmentof the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res.57:4593-4599 (1997)). The Fab of interest is then incubated overnight;however, the incubation may continue for a longer period (e.g., about 65hours) to ensure that equilibrium is reached. Thereafter, the mixturesare transferred to the capture plate for incubation at room temperature(e.g., for one hour). The solution is then removed and the plate washedeight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plateshave dried, 150 μl/well of scintillant (MICROSCINT-20™; Packard) isadded, and the plates are counted on a TOPCOUNT™ gamma counter (Packard)for ten minutes. Concentrations of each Fab that give less than or equalto 20% of maximal binding are chosen for use in competitive bindingassays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIAcore,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Stability

The invention provides antibodies with enhanced stability, for example,as compared to an anti-VEGF antibody, for instance, G6.31 (see, e.g.,U.S. Pat. No. 7,758,859 and International Application Pub. No. WO2005/012359, which are incorporated herein by reference in theirentirety). The stability of an antibody may be determined using anymethod known in the art, for example, differential scanning fluorimetry(DSF), circular dichroism (CD), intrinsic protein fluorescence,differential scanning calorimetry, spectroscopy, light scattering (e.g.,dynamic light scattering (DLS) and static light scattering (SLS),self-interaction chromatography (SIC). The anti-VEGF antibody may have,for example, an enhanced melting temperature (T_(m)), temperature ofaggregation (T_(agg)), or other metrics of stability compared to ananti-VEGF antibody, for example, G6.31.

In certain embodiments, an antibody provided herein has a T_(m) that isgreater than or equal to about 80° C. (e.g., about 81° C., about 82° C.,about 83° C., about 84° C., about 85° C., about 86° C., about 87° C.,about 88° C., about 89° C., about 90° C., about 91° C., about 92° C., orabout 93° C.). For example, in some instances, the anti-VEGF antibodyhas a T_(m) that is greater than or equal to about 83.5° C. (e.g., about83.5° C., about 84° C., about 85° C., about 86° C., about 87° C., about88° C., about 89° C., about 90° C., about 91° C., about 92° C., or about93° C.). In some instances, the anti-VEGF antibody has a T_(m) of about82° C. to about 92° C. (e.g., about 82° C., about 83° C., about 84° C.,about 85° C., about 86° C., about 87° C., about 88° C., about 89° C.,about 90° C., about 91° C., or about 92° C.). In some about instances,the anti-VEGF antibody has a T_(m) of about 82° C. In some instances,any of the preceding T_(m) values of an anti-VEGF antibody is determinedusing DSF. In some embodiments, the T_(m) value of an anti-VEGF antibodyis determined as described herein, for example, in Example 1.

3. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab-C, Fab′-SH, F(ab)₂, Fv, and scFv fragments, and other fragmentsdescribed below. For a review of certain antibody fragments, see Hudsonet al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see,e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

4. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, for example, inU.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA. 81:6851-6855 (1984). In one example, a chimeric antibody comprisesa non-human variable region (e.g., a variable domain derived from amouse, rat, hamster, rabbit, or non-human primate, such as a monkey) anda human constant domain. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, for example, CDRs, (or portionsthereof) are derived from a non-human antibody, and FRs (or portionsthereof) are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(e.g., the antibody from which the HVR residues are derived), e.g., torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008),and are further described, for example, in Riechmann et al., Nature332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321,and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describingspecificity determining region (SDR) grafting); Padlan, Mol. Immunol.28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal., J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

5. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology: U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

6. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

7. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In certain embodiments, one of the bindingspecificities is for VEGF and the other is for any other antigen (e.g.,a second biological molecule, e.g., interleukin-1 beta (IL-10),interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13(IL-13); IL-13 receptor (IL-13R); PDGF (e.g., PDGF-BB); angiopoietin;angiopoietin 2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-boundVEGF-receptor (mbVEGFR), or soluble VEGF receptor (sVEGFR)); ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk, such as complement pathway components C2,factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3;HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. Accordingly, the bispecific antibody may have bindingspecificity for VEGF and IL-1β; VEGF and IL-6; VEGF and IL-6R; VEGF andIL-13; VEGF and IL-13R; VEGF and PDGF (e.g., PDGF-BB); VEGF andangiopoietin; VEGF and Ang2; VEGF and Tie2; VEGF and S1P; VEGF andintegrin αvβ3; VEGF and integrin αvβ5; VEGF and integrin α5β1; VEGF andbetacellulin; VEGF and apelin/APJ; VEGF and erythropoietin; VEGF andcomplement factor D; VEGF and TNFα; VEGF and HtrA1; VEGF and a VEGFreceptor (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR); VEGF andST-2 receptor; VEGF and C2; VEGF and factor B; VEGF and factor H; VEGFand CFHR3; VEGF and C3b; VEGF and C5; VEGF and C5a; VEGF and C3a; VEGFand ARMS2; VEGF and TIMP3; VEGF and HLA; VEGF and IL-8; VEGF and CX3CR1;VEGF and TLR3; VEGF and TLR4; VEGF and CETP; VEGF and LIPC; VEGF andCOL10A1; or VEGF and TNFRSF10A. In certain embodiments, bispecificantibodies may bind to two different epitopes of VEGF. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress VEGF. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments (e.g., Fab, Fab′, or Fab-C fragments).

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-angiopoeitin 2 (Ang2) antibody disclosed in U.S. PatentApplication No. US 2014/0017244, which is incorporated herein byreference in its entirety. For example, the anti-VEGF/anti-Ang2bispecific antibody may include a first binding domain that binds VEGF(such as any of the anti-VEGF antibodies described herein) and a secondbinding domain that binds Ang2 that includes (a) an HVR-H1 comprisingthe amino acid sequence of GYYMH (SEQ ID NO: 62); (b) an HVR-H2comprising the amino acid sequence of WINPNSGGTNYAQKFQG (SEQ ID NO: 63);(c) an HVR-H3 comprising the amino acid sequence of SPNPYYYDSSGYYYPGAFDI(SEQ ID NO: 64); (d) an HVR-L1 comprising the amino acid sequence ofGGNNIGSKSVH (SEQ ID NO: 65); (e) an HVR-L2 comprising the amino acidsequence of DDSDRPS (SEQ ID NO: 66); and (t) an HVR-L3 comprising theamino acid sequence of QVWDSSSDHWV (SEQ ID NO: 67), or a combination ofone or more of the above HVRs and one or more variants thereof having atleast about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to any one of SEQ ID NOs: 62-67.

In some instances, the anti-VEGF/anti-Ang2 bispecific antibody mayinclude a first binding domain that binds VEGF (such as any of theanti-VEGF antibodies described herein) and a second binding domain thatbinds to Ang2 and includes (a) a VH domain comprising an amino acidsequence having at least 80% sequence identity (e.g., 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:68; (b) a VL domain comprising an amino acid sequence having at least80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 69; or (c) a VH domain asin (a) and a VL domain as in (b). In some instances, theanti-VEGF/anti-Ang2 bispecific antibody may include a first bindingdomain that binds VEGF (such as any of the anti-VEGF antibodiesdescribed herein) and a second binding domain that specifically bind toAng2, wherein the second binding domain is any antibody binding domaindescribed in International Patent Application Publication No. WO2010/069532, which is incorporated herein by reference in its entirety,or a variant thereof.

In other instances, the anti-VEGF/anti-Ang2 bispecific antibody is anyanti-VEGF/anti-Ang2 bispecific antibody described in InternationalPatent Application Publication No. WO 2016/073157.

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-IL-6 antibody. In some instances, an anti-VEGF/anti-IL-6bispecific antibody may include a first binding domain that binds VEGF(such as any of the anti-VEGF antibodies described herein) and a secondbinding domain that binds IL-6. The second binding domain may be abinding domain of any anti-IL-6 antibody known in the art, for example,EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890, which isincorporated herein by reference in its entirety), siltuximab(SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117. PF-04236921, or a variant thereof.

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-IL-6R antibody. In some instances, ananti-VEGF/anti-IL-6R bispecific antibody may include a first bindingdomain that binds VEGF (such as any of the anti-VEGF antibodiesdescribed herein) and a second binding domain that binds IL-6R. Thesecond binding domain may be a binding domain any anti-IL-6R antibodyknown in the art, for example, tocilizumab (ACTEMRA®) (see, e.g., WO1992/019579, which is incorporated herein by reference in its entirety),sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, for example, in Tutt etal., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to VEGF as well asanother, different antigen (see, e.g., US 2008/0069820).

8. Antibody Variants

In certain embodiments, amino acid sequence variants (e.g., antibodyvariants including one or more amino acid residue alterations) of theantibodies provided herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of an antibodymay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example, antigenbinding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Preferred Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues and/or FR residues of a parent antibody(e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further study will have modifications (e.g.,improvements) in certain biological properties (e.g., increasedaffinity, increased stability, increased expression, altered pI, and/orreduced immunogenicity) relative to the parent antibody and/or will havesubstantially retained certain biological properties of the parentantibody. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated, for example, using phagedisplay-based affinity maturation techniques such as those describedherein. Briefly, one or more HVR residues are mutated and the variantantibodies displayed on phage and screened for a particular biologicalactivity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues thatcontact antigen, with the resulting variant VH or VL being tested forbinding affinity. Affinity maturation by constructing and reselectingfrom secondary libraries has been described, for example, in Hoogenboomet al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed.,Human Press, Totowa, N.J., (2001)). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more FRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Suchalterations may, for example, improve antibody affinity and/or stability(e.g., as assessed by an increased melting temperature).

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, for example, US Patent Publication Nos. US 2003/0157108; US2004/0093621. Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al., J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat ApplNo US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, for example, in WO1997/30087; WO 1998/58964; and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid residue alteration (e.g., asubstitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invive cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337; and Bruggemann et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCYTOTOX 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, for example, in a animal model such as that disclosedin Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1qbinding assays may also be carried out to confirm that the antibody isunable to bind C1q and hence lacks CDC activity. See, for example, C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, e.g.,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg etal., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance/half life determinations canalso be performed using methods known in the art (see, e.g., Petkova etal., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), for example, as described inU.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,and the like. Additional antibody conjugates are described herein, forexample, in Section K below and in Example 13.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

f) Isoelectric Point Variants

The invention provides antibodies variants with altered isoelectricpoints. For example, the invention provides antibodies variants with areduced isoelectric point (pI), for example, as compared to an anti-VEGFantibody, for instance, G6.31. In some instances, the surface charge isreduced at physiological pH. In some instances, the anti-VEGF antibodyhas a pI equal to or lower than about 8 (e.g., about 8, about 7, about6, about 5, or about 4). In some instances, the antibody has a pI fromabout 4 to about 8 (e.g., about 4, about 5, about 6, about 7, or about8). In some instances, the anti-VEGF antibody has a pI from about 5 toabout 7 (e.g., about 5, about 6, or about 7). In some instances, theanti-VEGF antibody has a pI from about 5 to about 6 (e.g., about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, or about 6).

Antibodies of the invention may be engineered to have a reduced pI, forexample, by substituting wild-type amino acid residues at a givenposition with an amino acid having a lower pI. The pI of an amino acidcan be determined based on the pKa values of the amine (—NH₂),carboxylic acid (—COOH), and side-chain of the amino acid, which areknown in the art. In some embodiments, surface-exposed amino acidresidues may be substituted to reduce the pI of an antibody. In oneembodiment, surface-exposed amino acid residues may be substituted withglutamate (E). In one embodiment, surface-exposed amino acid residuesmay be substituted with aspartate (D).

B. Recombinant Methods and Compositions

Any of the antibodies (e.g., anti-VEGF antibodies) described herein maybe produced using recombinant methods and compositions, for example, asdescribed in U.S. Pat. No. 4,816,567. In one embodiment, an isolatednucleic acid encoding an anti-VEGF antibody described herein isprovided. Such a nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such a nucleic acid are provided. In a further embodiment, ahost cell comprising such a nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method ofmaking an anti-VEGF antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-VEGF antibody, nucleic acidencoding an antibody, for example, as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, for example, U.S.Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. See also Chariton,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli. After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, for example,U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)):baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, for example, Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003).

C. Assays

Anti-VEGF antibodies provided herein, as well as compositions thatinclude anti-VEGF antibodies (e.g., any anti-VEGF antibody providedherein), such as antibody conjugates, fusion proteins, and polymericformulations, may be identified, screened for, or characterized fortheir physical/chemical properties and/or biological activities byvarious assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention, or an antibody conjugate,fusion protein, or polymeric formulation thereof, is tested for itsantigen binding activity, e.g., by known methods such as ELISA, Westernblot, etc.

In another aspect, competition assays may be used to identify anantibody that competes with an antibody as described herein, or anantibody conjugate, fusion protein, or polymeric formulation thereof,for binding to VEGF. In certain embodiments, such a competing antibodybinds to the same epitope (e.g., a linear or a conformational epitope)that is bound by an antibody as described herein. Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols,” in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized VEGF is incubated in asolution comprising a first labeled antibody that binds to VEGF and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to VEGF. The second antibodymay be present in a hybridoma supernatant. As a control, immobilizedVEGF is incubated in a solution comprising the first labeled antibodybut not the second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to VEGF, excess unboundantibody is removed, and the amount of label associated with immobilizedVEGF is measured. If the amount of label associated with immobilizedVEGF is substantially reduced in the test sample relative to the controlsample, then that indicates that the second antibody is competing withthe first antibody for binding to VEGF. See Harlow and Lane (1988)Antibodies: A Laboratory Manual ch, 14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-VEGF antibodies,or antibody conjugates, fusion proteins, or polymeric formulationsthereof, having biological activity. Biological activity may include,for example, binding to VEGF (e.g., VEGF in the blood stream), or apeptide fragment thereof, either in vivo, in vitro, or ex vivo. Incertain embodiments, biological activity may include blocking orneutralizing VEGF, or preventing VEGF from binding to a ligand, forexample, a receptor such as KDR or Flt-1. Antibodies, or antibodyconjugates, fusion proteins, or polymeric formulations thereof, havingsuch biological activity in vivo and/or in vitro are also provided. Incertain embodiments, an antibody of the invention, or an antibodyconjugate, fusion protein, or polymeric formulation thereof, is testedfor such biological activity.

3. Stability Assays

In one aspect, assays are provided for determining the stability (e.g.,thermostability) of an anti-VEGF antibody, or an antibody conjugate,fusion protein, or polymeric formulation thereof. For example, thestability of an antibody, or an antibody conjugate, fusion protein, orpolymeric formulation thereof, may be determined using any method knownin the art, for example, differential scanning fluorimetry (DSF),circular dichroism (CD), intrinsic protein fluorescence, differentialscanning calorimetry, spectroscopy, light scattering (e.g., dynamiclight scattering (DLS) and static light scattering (SLS),self-interaction chromatography (SIC). The stability of an assay may bedetermined as described herein, for example, using DSF as described, forexample, in Examples 1 and 2.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-VEGFantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296: Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, cumin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Affinity Purification, Diagnostics, andDetection

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a SEPHADEX® resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theantigen to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the antigen to be purified, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theantigen from the antibody.

In certain embodiments, any of the anti-VEGF antibodies provided hereinis useful for detecting the presence of VEGF in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue, such as blood (e.g., whole blood, plasma, and/or serum),tissue samples (e.g., tumor samples), and the like.

In one embodiment, an anti-VEGF antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of VEGF in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-VEGF antibody as described herein under conditionspermissive for binding of the anti-VEGF antibody to VEGF, and detectingwhether a complex is formed between the anti-VEGF antibody and VEGF.Such method may be an in vitro or in vivo method. In one embodiment, ananti-VEGF antibody is used to select subjects eligible for therapy withan anti-VEGF antibody, for example, where VEGF is a biomarker forselection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include disorders associated with pathological angiogenesis(e.g., ocular disorders and cell proliferative disorders) and/ordisorders associated with undesirable vascular permeability (e.g., edemaassociated with brain tumors, ascites associated with malignancies,Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardialeffusion, pleural effusion, and permeability associated withcardiovascular diseases). In some instances, the ocular disorder is AMD(e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographicatrophy (GA)), macular degeneration, macular edema, DME (e.g., focal,non-center DME or diffuse, center-involved DME), retinopathy, diabeticretinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR(NPDR), or high-altitude DR), other ischemia-related retinopathies, ROP,retinal vein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO)forms), CNV (e.g., myopic CNV), corneal neovascularization, diseasesassociated with corneal neovascularization, retinal neovascularization,diseases associated with retinal/choroidal neovascularization,pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye,FEVR, Coats' disease, Nome Disease, OPPG, subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis (e.g.,infectious conjunctivitis and non-infectious (e.g, allergic)conjunctivitis), Leber congenital amaurosis (also known as Leber'scongenital amaurosis or LCA), uveitis (including infectious andnon-infectious uveitis), choroiditis (e.g., multifocal choroiditis),ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, orSjögren's disease. In some instances, the ocular disorder is retinopathyincluding proliferative diabetic retinopathy, choroidalneovascularization (CNV), age-related macular degeneration (AMD),diabetic and other ischemia-related retinopathies, diabetic macularedema (DME), pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (including central(CRVO) and branched (BRVO) forms), corneal neovascularization, retinalneovascularization, retinopathy of prematurity (ROP), familial exudativevitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,or hypertensive retinopathy. In some instances, the cell proliferativedisorder is cancer. In some instances, the cancer is breast cancer,colorectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma(NHL), renal cancer, prostate cancer, liver cancer, head and neckcancer, melanoma, ovarian cancer, mesothelioma, or multiple myeloma.

In certain embodiments, labeled anti-VEGF antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, 3-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyze for binding with a limited amountof antibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyze that are boundto the antibodies may conveniently be separated from the standard andanalyze which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, for example, U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) or a dye so that the tumor can belocalized using immunoscintiography.

F. Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

G. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-VEGF antibody as describedherein. or an antibody conjugate, fusion protein, or polymericformulation thereof. are prepared by mixing such antibody having thedesired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rtHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, for example, byfiltration through sterile filtration membranes.

In certain embodiments, the pharmaceutical formulation includes one ormore additional compounds. In certain embodiments, the additionalcompound binds to a second biological molecule selected from the groupconsisting of IL-1β, IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin;angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin;apelin/APJ; erythropoietin; complement factor D; TNFα; HtrA1; a VEGFreceptor; ST-2 receptor; and proteins genetically linked to age-relatedmacular degeneration (AMD) risk, such as complement pathway componentsC2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2;TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof.

For example, in some instances, the additional compound is a bispecificantibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody, such asRG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecific antibodydisclosed in WO 2010/069532 or WO 2016/073157.

In another example, in some instances, the additional compound is ananti-IL-6 antibody, for example, EBI-031 (Eleven Biotherapeutics; see,e.g., WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clazakizumab,sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921,or a variant thereof.

In a still further example, in some instances, the additional compoundis an anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see,e.g., WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

H. Therapeutic Methods and Compositions

Any of the anti-VEGF antibodies, antibody conjugates (e.g., HAconjugates. PEG conjugates, and prodrug antibody conjugates), fusionproteins, and polymeric formulations provided herein may be used intherapeutic methods.

In one aspect, an anti-VEGF antibody for use as a medicament isprovided. In another aspect, an antibody conjugate for use as amedicament is provided. In yet another aspect, a fusion protein for useas a medicament is provided. In a still further aspect, a polymericformulation for use as a medicament is provided. In further aspects, theinvention provides an anti-VEGF antibody for use in treating a disorderassociated with pathological angiogenesis. In another aspect, theinvention provides an antibody conjugate for use in treating a disorderassociated with pathological angiogenesis. In yet other aspects, theinvention provides a fusion protein for use in treating a disorderassociated with pathological angiogenesis. In other aspects, theinvention provides a polymeric formulation for use in treating adisorder associated with pathogical angiogenesis. In some embodiments,the disorder associated with pathological angiogenesis is an oculardisorder or a cell proliferative disorder. In some instances, the oculardisorder is AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD,or geographic atrophy (GA)), macular degeneration, macular edema, DME(e.g., focal, non-center DME or diffuse, center-involved DME),retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR),non-proliferative DR (NPDR), or high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g, allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis,blepharitis, dry eye, traumatic eye injury, or Sjögren's disease. Insome instances, the cell proliferative disorder is cancer. In someinstances, the cancer is breast cancer, colorectal cancer, non-smallcell lung cancer, non-Hodgkins lymphoma (NHL), renal cancer, prostatecancer, liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, or multiple myeloma. In another aspect, an anti-VEGFantibody for use in treating a disorder associated with undesirablevascular permeability is provided. In some instances, the disorderassociated with undesirable vascular permeability is edema associatedwith brain tumors, ascites associated with malignancies, Meigs'syndrome, lung inflammation, nephrotic syndrome, pericardial effusion,pleural effusion, or permeability associated with cardiovasculardiseases.

In another aspect, an anti-VEGF antibody for use in a method oftreatment is provided. In another aspect, an antibody conjugate for usein a method of treatment is provided. In yet another aspect, a fusionprotein for use in a method of treatment is provided. In a still furtheraspect, a polymeric formulation for use in a method of treatment isprovided. In certain instances, the invention provides an anti-VEGFantibody for use in a method of treating a subject having a disorderassociated with pathological angiogenesis comprising administering tothe individual an effective amount of the anti-VEGF antibody. Theinvention also provides an antibody conjugate for use in a method oftreating a subject having a disorder associated with pathologicalangiogenesis comprising administering to the individual an effectiveamount of the antibody conjugate. The invention also provides a fusionprotein for use in a method of treating a subject having a disorderassociated with pathological angiogenesis comprising administering tothe individual an effective amount of the fusion protein. The inventionalso provides a polymeric formulation for use in a method of treating asubject having a disorder associated with pathological angiogenesiscomprising administering to the individual an effective amount of thepolymeric formulation. In some instances, the ocular disorder is AMD(e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographicatrophy (GA)), macular degeneration, macular edema, DME (e.g., focal,non-center DME or diffuse, center-involved DME), retinopathy, diabeticretinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR(NPDR), or high-altitude DR), other ischemia-related retinopathies, ROP,retinal vein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO)forms), CNV (e.g., myopic CNV), corneal neovascularization, diseasesassociated with corneal neovascularization, retinal neovascularization,diseases associated with retinal/choroidal neovascularization,pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye,FEVR, Coats' disease, Norrie Disease, OPPG, subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis (e.g.,infectious conjunctivitis and non-infectious (e.g, allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis (e.g., multifocalchoroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, or Sjögren's disease. In some instances, the cell proliferativedisorder is cancer. In some instances, the cancer is breast cancer,colorectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma(NHL), renal cancer, prostate cancer, liver cancer, head and neckcancer, melanoma, ovarian cancer, mesothelioma, or multiple myeloma.

In other instances, the invention provides an anti-VEGF antibody for usein a method of treating an individual having a disorder associated withundesirable vascular permeability. In some instances, the disorderassociated with undesirable vascular permeability is edema associatedwith brain tumors, ascites associated with malignancies, Meigs'syndrome, lung inflammation, nephrotic syndrome, pericardial effusion,pleural effusion, or permeability associated with cardiovasculardiseases. Any of the preceding uses may further include administering tothe individual an effective amount of at least one additionaltherapeutic agent, for example, as described below.

In some instances, the invention provides an anti-VEGF antibody for usein reducing or inhibiting angiogenesis in a subject. In another aspect,an antibody conjugate for use in reducing or inhibiting angiogenesis ina subject is provided. In yet another aspect, a fusion protein for usein reducing or inhibiting angiogenesis in a subject is provided. In astill further aspect, a polymeric formulation for use in reducing orinhibiting angiogenesis in a subject is provided. In certainembodiments, the invention provides an anti-VEGF antibody for use in amethod of reducing or inhibiting angiogenesis in a subject comprisingadministering to the individual an effective of the anti-VEGF antibodyto reduce or inhibit angiogenesis. The invention also provides anantibody conjugate for use in a method of reducing or inhibitingangiogenesis in a subject comprising administering to the individual aneffective amount of the antibody conjugate. The invention also providesa fusion protein for use in a method of reducing or inhibitingangiogenesis in a subject comprising administering to the individual aneffective amount of the fusion protein. The invention also provides apolymeric formulation for use in a method of reducing or inhibitingangiogenesis in a subject comprising administering to the individual aneffective amount of the polymeric formulation. In other instances, theinvention provides an anti-VEGF antibody for use in reducing orinhibiting vascular permeability in a subject. In certain embodiments,the invention provides an anti-VEGF antibody for use in a reducing orinhibiting vascular permeability in a subject comprising administeringto the individual an effective of the anti-VEGF antibody to reduce orinhibit vascular permeability. A “subject” according to any of the aboveuses may be a human.

In some instances, the invention provides an anti-VEGF antibody for usein treating an autoimmune disease in a subject. In some instances, theautoimmune disorder is rheumatoid arthritis, ankylosing spondylitis,psoriatic arthritis and Crohn's disease. Any of the preceding uses mayfurther include administering to the individual an effective amount ofat least one additional therapeutic agent, for example, as describedbelow.

The invention provides for the use of an anti-VEGF antibody in themanufacture or preparation of a medicament. The invention also providesfor the use of an antibody conjugate in the manufacture or preparationof a medicament. Further still, the invention provides for the use of afusion protein in the manufacture or preparation of a medicament. Theinvention also provides for the use of a polymeric formulation in themanufacture or preparation of a medicament. For example, in oneinstance, the medicament is for treatment of a disorder associated withpathological angiogenesis. In a further instance, the medicament is foruse in a method of treating a disorder associated with pathologicalangiogenesis comprising administering to a subject having a disorderassociated with pathological angiogenesis an effective amount of themedicament. In some instances, the ocular disorder is AMD (e.g., wetAMD, dry AMD, intermediate AMD, advanced AMD, or geographic atrophy(GA)), macular degeneration, macular edema, DME (e.g., focal, non-centerDME or diffuse, center-involved DME), retinopathy, diabetic retinopathy(DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), orhigh-altitude DR), other ischemia-related retinopathies, ROP, retinalvein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms),CNV (e.g., myopic CNV), corneal neovascularization, diseases associatedwith corneal neovascularization, retinal neovascularization, diseasesassociated with retinal/choroidal neovascularization, pathologic myopia,von Hippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats'disease, Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis,ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa(RP), hypertensive retinopathy, retinal angiomatous proliferation,macular telangiectasia, iris neovascularization, intraocularneovascularization, retinal degeneration, cystoid macular edema (CME),vasculitis, papilloedema, retinitis, conjunctivitis (e.g., infectiousconjunctivitis and non-infectious (e.g, allergic) conjunctivitis), Lebercongenital amaurosis, uveitis (including infectious and non-infectiousuveitis), choroiditis (e.g., multifocal choroiditis), ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, or Sjögren'sdisease. In some instances, the cell proliferative disorder is cancer.In some instances, the cancer is breast cancer, colorectal cancer,non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cancer,prostate cancer, liver cancer, head and neck cancer, melanoma, ovariancancer, mesothelioma, or multiple myeloma. In a further instance, themedicament is for reducing or inhibiting angiogenesis in a subject. In afurther instance, the medicament is for use in a method of reducing orinhibiting angiogenesis in a subject comprising administering to thesubject an amount effective of the medicament to reduce or inhibitangiogenesis. In any of the preceding uses of medicaments, the methodmay include administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In another instance, the medicament is for treatment of a disorderassociated with undesirable vascular permeability. In some instances,the disorder associated with undesirable vascular permeability is edemaassociated with brain tumors, ascites associated with malignancies,Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardialeffusion, pleural effusion, or permeability associated withcardiovascular diseases. In a further instance, the medicament is foruse in a method of treating a disorder associated with undesirablevascular permeability comprising administering to a subject having aassociated with undesirable vascular permeability an effective amount ofthe medicament. In another instance, the medicament is for reducing orinhibiting vascular permeability in a subject. In a further instance,the medicament is for use in a method of reducing or inhibiting vascularpermeability in a subject comprising administering to the subject anamount effective of the medicament to reduce or inhibit angiogenesis. Inany of the preceding uses of medicaments, the method may includeadministering to the subject an effective amount of at least oneadditional therapeutic agent, e.g., as described below. A “subject”according to any of the above uses may be a human.

In another instance, the medicament is for treatment of an autoimmunedisorder. In some instances, the autoimmune disorder is rheumatoidarthritis, ankylosing spondylitis, psoriatic arthritis and Crohn'sdisease. In a further instance, the medicament is for use in a method oftreating an autoimmune disorder comprising administering to a subjecthaving an autoimmune disorder an effective amount of the medicament. A“subject” according to any of the above uses may be a human.

The invention provides a method for treating a disorder associated withpathological angiogenesis. In one embodiment, the method comprisesadministering to an individual having a disorder associated withpathological angiogenesis an effective amount of an anti-VEGF antibody.In another example, the method comprises administering to an individualhaving a disorder associated with pathological angiogenesis an effectiveamount of an antibody conjugate. In yet another example, the methodcomprises administering to an individual having a disorder associatedwith pathological angiogenesis an effective amount of a fusion protein.In yet another example, the method comprises administering to anindividual having a disorder associated with pathological angiogenesisan effective amount of a polymeric formulation. In some instances, theocular disorder is AMD (e.g., wet AMD, dry AMD, intermediate AMD,advanced AMD, or geographic atrophy (GA)), macular degeneration, macularedema. DME (e.g., focal, non-center DME or diffuse, center-involvedDME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR(PDR), non-proliferative DR (NPDR), or high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g, allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis,blepharitis, dry eye, traumatic eye injury, or Sjögren's disease. Insome instances, the cell proliferative disorder is cancer. In someinstances, the cancer is breast cancer, colorectal cancer, non-smallcell lung cancer, non-Hodgkins lymphoma (NHL), renal cancer, prostatecancer, liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, or multiple myeloma. In further instances, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, as described below. A“subject” according to any of the above methods may be a human.

The invention provides a method for treating a disorder associated withundesirable vascular permeability. In one embodiment, the methodcomprises administering to an individual having a disorder associatedwith undesirable vascular permeability an effective amount of ananti-VEGF antibody. In some instances, the disorder associated withundesirable vascular permeability is edema associated with brain tumors,ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion, pleuraleffusion, or permeability associated with cardiovascular diseases. Infurther instances, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below. A “subject” according to any of the abovemethods may be a human.

It is contemplated that the antibody of the present invention, orantibody conjugate, fusion protein, or polymeric formulation thereof,may be used to treat a mammal. In one embodiment, the antibody, orantibody conjugate, fusion protein, or polymeric formulation thereof, isadministered to a nonhuman mammal for the purposes of obtainingpreclinical data, for example. Exemplary nonhuman mammals to be treatedinclude nonhuman primates, dogs, cats, rodents (e.g., mice and rats) andother mammals in which preclinical studies are performed. Such mammalsmay be established animal models for a disease to be treated with theantibody or may be used to study toxicity or pharmacokinetics of theantibody of interest. In each of these embodiments, dose escalationstudies may be performed in the mammal. The antibody may be administeredto a host rodent in a solid tumor model, for example. The antibody, orantibody conjugate, fusion protein, or polymeric formulation thereof,may be administered to a host (e.g., a rodent, e.g., a rabbit) forocular pharmacokinetic studies, for example, by intravitrealadministration (e.g., intravitreal injection) or using a port deliverydevice.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-VEGF antibodies, antibody conjugates, fusionproteins, and/or polymeric formulations provided herein, for example,for use in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-VEGF antibodies,antibody conjugates, fusion proteins, and/or polymeric formulationsprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-VEGFantibodies, antibody conjugates, fusion proteins, and/or polymericformulations provided herein and at least one additional therapeuticagent, for example, as described below. In certain embodiments, thepharmaceutical formulation comprises one or more additional compounds.In certain embodiments, the additional compound binds to a secondbiological molecule selected from the group consisting of IL-1β; IL-6;IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrinsαvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin;complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; andproteins genetically linked to age-related macular degeneration (AMD)risk, such as complement pathway components C2, factor B, factor H,CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8(IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC, COL10A1; and TNFRSF10A. Incertain embodiments, the additional compound is an antibody orantigen-binding fragment thereof. For example, in some instances, theadditional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

Antibodies of the invention, or antibody conjugates, fusion proteins, orpolymeric formulations thereof, can be used either alone or incombination with other agents in a therapy. For instance, an antibody ofthe invention, or an antibody conjugate, fusion protein, and/orpolymeric formulation thereof, may be co-administered with at least oneadditional therapeutic agent. In certain embodiments, an additionaltherapeutic agent is another antibody, a chemotherapeutic agent, acytotoxic agent, an anti-angiogenic agent, an immunosuppressive agent, aprodrug, a cytokine, a cytokine antagonist, cytotoxic radiotherapy, acorticosteroid, an anti-emetic, a cancer vaccine, an analgesic, agrowth-inhibitory agent, or combinations thereof.

For example, in certain embodiments, any of the preceding methodsfurther comprises administering one or more additional compounds. Incertain embodiments, the anti-VEGF antibody, antibody conjugate, fusionprotein, or polymeric formulation is administered simultaneously withthe additional compound(s). In certain embodiments, the anti-VEGFantibody, antibody conjugate, fusion protein, or polymeric formulationis administered before or after the additional compound(s). In certainembodiments, the additional compound binds to a second biologicalmolecule selected from the group consisting of IL-1β; IL-6; IL-6R;IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrins αvβ3,αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complementfactor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and proteinsgenetically linked to AMD risk, such as complement pathway componentsC2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2;TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC;COL10A1; and TNFRSF10A. In certain embodiments, the additional compoundis an antibody or antigen-binding fragment thereof. In certainembodiments according to (or as applied to) any of the embodimentsabove, the ocular disorder is an intraocular neovascular diseaseselected from the group consisting of proliferative retinopathies,choroidal neovascularization (CNV), age-related macular degeneration(AMD), diabetic and other ischemia-related retinopathies, diabeticmacular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), including CRVOand BRVO, corneal neovascularization, retinal neovascularization, andretinopathy of prematurity (ROP). For example, in some instances, theadditional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

In some instances, an antibody of the invention, or an antibodyconjugate, fusion protein, and/or polymeric formulation thereof, may beadministered in combination with at least one additional therapeuticagent for treatment of an ocular disorder, for example, an oculardisorder described herein (e.g., AMD (e.g., wet AMD), DME, DR, or RVO).Exemplary additional therapeutic agents for combination therapy fortreatment of ocular disorders include, without limitation,anti-angiogenic agents, such as VEGF antagonists, including, forexample, anti-VEGF antibodies (e.g., the anti-VEGF Fab LUCENTIS®(ranibizumab)), soluble receptor fusion proteins (e.g., the recombinantsoluble receptor fusion protein EYLEA® (aflibercept, also known as VEGFTrap Eye; Regeneron/Aventis)), aptamers (e.g., the anti-VEGF pegylatedaptamer MACUGEN® (pegaptanib sodium; NeXstar Pharmaceuticals/OSIPharmaceuticals)), and VEGFR tyrosine kinase inhibitors (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)); Tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE®(anecortave acetate for depot suspension; Alcon, Inc.); CombretastatinA4 Prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenontriamcinolone acetonide; intravitreal crystalline triamcinoloneacetonide; matrix metalloproteinase inhibitors (e.g., Prinomastat(AG3340; Pfizer)); fluocinolone acetonide (including fluocinoloneintraocular implant; Bausch & Lomb/Control Delivery Systems); linomide;inhibitors of integrin 133 function; angiostatin, and combinationsthereof. These and other therapeutic agents that can be administered incombination with an antibody of the invention are described, forexample, in U.S. Patent Application No. US 2014/0017244, which isincorporated herein by reference in its entirety.

Further examples of additional therapeutic agents that can be used incombination with an antibody of the invention, or an antibody conjugate,fusion protein, and/or polymeric formulation thereof, for treatment ofan ocular disorder (e.g., AMD, DME, DR, or RVO), include, but are notlimited to, VISUDYNE® (verteporfin; a light-activated drug that istypically used in conjunction with photodynamic therapy with anon-thermal laser), PKC412, Endovion (NS 3728; NeuroSearch A/S),neurotrophic factors (e.g., glial derived neurotrophic factor (GDNF) andciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®,9-cis-retinal, eye medication (e.g., phospholine iodide, echothiophate,or carbonic anhydrase inhibitors), veovastat (AE-941; AEternaLaboratories, Inc.), Sirna-027 (AGF-745; Sirna Therapeutics, Inc.),neurotrophins (including, by way of example only, NT-4/5, Genentech),Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals),integrin antagonists (including those from Jerini AG and AbbottLaboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.),thalidomide (as used, for example, by EntreMed, Inc.), cardiotrophin-1(Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (TorayIndustries), NTC-200 (Neurotech), tetrathiomolybdate (University ofMichigan), LYN-002 (Lynkeus Biotech), microalgal compound(Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc),ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosinekinase inhibitors (e.g., those from Allergan, SUGEN, or Pfizer),NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS FranceSA), retinal cell ganglion neuroprotectants (Cogent Neurosciences),N-nitropyrazole derivatives (Texas A&M University System), KP-102(Krenitsky Pharmaceuticals), cyclosporin A, therapeutic agents used inphotodynamic therapy (e.g., VISUDYNE®; receptor-targeted PDT,Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT;verteporfin, QLT Inc.; rostaporfin with PDT, Miravent MedicalTechnologies; talaporfin sodium with PDT, Nippon Petroleum; andmotexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides(including, by way of example, products tested by Novagali Pharma SA andISIS-13650, Isis Pharmaceuticals), and combinations thereof.

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, may be administered in combinationwith a therapy or surgical procedure for treatment of an ocular disorder(e.g., AMD, DME, DR, or RVO), including, for example, laserphotocoagulation (e.g., panretinal photocoagulation (PRP)), drusenlasering, macular hole surgery, macular translocation surgery,implantable miniature telescopes, PHI-motion angiography (also known asmicro-laser therapy and feeder vessel treatment), proton beam therapy,microstimulation therapy, retinal detachment and vitreous surgery,scleral buckle, submacular surgery, transpupillary thermotherapy,photosystem I therapy, use of RNA interference (RNAi), extracorporealrheopheresis (also known as membrane differential filtration andrheotherapy), microchip implantation, stem cell therapy, genereplacement therapy, ribozyme gene therapy (including gene therapy forhypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEFgene therapy, GenVec), photoreceptor/retinal cells transplantation(including transplantable retinal epithelial cells, Diacrin, Inc.;retinal cell transplant, Cell Genesys, Inc.), acupuncture, andcombinations thereof.

In some instances, an antibody of the invention, or an antibodyconjugate, fusion protein, and/or polymeric formulation thereof, can beadministered in combination with an anti-angiogenic agent for treatmentof an ocular disorder (e.g., AMD, DME, DR, or RVO). Any suitableanti-angiogenic agent can be used in combination with an antibody of theinvention, including, but not limited to, those listed by Carmeliet etal. Nature 407:249-257, 2000. In some embodiments, the anti-angiogenicagent is a VEGF antagonist, including, but not limited to, an anti-VEGFantibody (e.g., the anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258(formerly ESBA-1008, an anti-VEGF single-chain antibody fragment;Novartis), or a bispecific anti-VEGF antibody (e.g., ananti-VEGF/anti-angiopoeitin 2 bispecific antibody such as RG-7716;Roche)), a soluble recombinant receptor fusion protein (e.g., EYLEA®(aflibercept)), a VEGF variant, a soluble VEGFR fragment, an aptamercapable of blocking VEGF (e.g., pegaptanib) or VEGFR, a neutralizinganti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosinekinases, an anti-VEGF DARPin® (e.g., abicipar pegol), a smallinterfering RNAs which inhibits expression of VEGF or VEGFR, a VEGFRtyrosine kinase inhibitor (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)), and combinations thereof. In some instances, thebispecific anti-VEGF antibody binds to a second biological molecule,including but not limited to IL-1β; IL-6; IL-6R; PDGF (e.g., PDGF-BB);angiopoietin; angiopoietin 2; Tie2; SIP; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, orsVEGFR); ST-2 receptor; and proteins genetically linked to age-relatedmacular degeneration (AMD) risk, such as complement pathway componentsC2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2;TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. For example, in some instances, the additional compound is abispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody,such as RG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecificantibody disclosed in WO 2010/069532 or WO 2016/073157 or a variantthereof.

Other suitable anti-angiogenic agents that may be administered incombination with an antibody of the invention, or an antibody conjugate,fusion protein, and/or polymeric formulation thereof, for treatment ofan ocular disorder (e.g., AMD, DME, DR, or RVO) include corticosteroids,angiostatic steroids, anecortave acetate, angiostatin, endostatin,tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors,insulin-like growth factor-binding protein 3 (IGFBP3), stromal derivedfactor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigmentepithelium-derived factor (PEDF), gamma-secretase, Delta-like ligand 4,integrin antagonists, hypoxia-inducible factor (HIF)-1α antagonists,protein kinase CK2 antagonists, agents that inhibit stem cell (e.g.,endothelial progenitor cell) homing to the site of neovascularization(e.g., an anti-vascular endothelial cadherin (CD-144) antibody and/or ananti-SDF-1 antibody), and combinations thereof.

In a further example, in some instances, an antibody of the invention,or an antibody conjugate, fusion protein, and/or polymeric formulationthereof, can be administered in combination with an agent that hasactivity against neovascularization for treatment of an ocular disorder(e.g., AMD, DME, DR, or RVO), such as an anti-inflammatory drug, amammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin,AFINITOR® (everolimus), and TORISEL® (temsirolimus)), cyclosporine, atumor necrosis factor (TNF) antagonist (e.g., an anti-TNFα antibody orantigen-binding fragment thereof (e.g., infliximab, adalimumab,certolizumab pegol, and golimumab) or a soluble receptor fusion protein(e.g., etanercept)), an anti-complement agent, a nonsteroidalantiinflammatory agent (NSAID), or combinations thereof.

In a still further example, in some instances, an antibody of theinvention, or an antibody conjugate, fusion protein, and/or polymericformulation thereof, can be administered in combination with an agentthat is neuroprotective and can potentially reduce the progression ofdry AMD to wet AMD, such as the class of drugs called the“neurosteroids,” which include drugs such as dehydroepiandrosterone(DHEA) (brand names: PRASTERA™ and FIDELIN®), dehydroepiandrosteronesulfate, and pregnenoione sulfate.

Any suitable AMD therapeutic agent can be administered as an additionaltherapeutic agent in combination with an antibody of the invention, oran antibody conjugate, fusion protein, and/or polymeric formulationthereof, for treatment of an ocular disorder (e.g., AMD, DME, DR, orRVO), including, but not limited to, a VEGF antagonist, for example, ananti-VEGF antibody (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerlyESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), or abispecific anti-VEGF antibody (e.g., an anti-VEGF/anti-angiopoeitin 2bispecific antibody such as RG-7716; Roche)), a soluble VEGF receptorfusion protein (e.g., EYLEA® (aflibercept)), an anti-VEGF DARPin® (e.g.,abicipar pegol; Molecular Partners AG/Allergan), or an anti-VEGF aptamer(e.g, MACUGEN® (pegaptanib sodium)); a platelet-derived growth factor(PDGF) antagonist, for example, an anti-PDGF antibody, an anti-PDGFRantibody (e.g., REGN2176-3), an anti-PDGF-BB pegylated aptamer (e.g.,FOVISTA®; Ophthotech/Novartis), a soluble PDGFR receptor fusion protein,or a dual PDGF/VEGF antagonist (e.g., a small molecule inhibitor (e.g.,DE-120 (Santen) or X-82 (TyrogeneX)) or a bispecific anti-PDGF/anti-VEGFantibody)); VISUDYNE® (verteporfin) in combination with photodynamictherapy; an antioxidant; a complement system antagonist, for example, acomplement factor C5 antagonist (e.g., a small molecule inhitor (e.g.,ARC-1905; Opthotech) or an anti-C5 antibody (e.g., LFG-316; Novartis), aproperdin antagonist (e.g., an anti-properdin antibody, e.g., CLG-561;Alcon), or a complement factor D antagonist (e.g., an anti-complementfactor D antibody, e.g, lampalizumab; Roche)); a visual cycle modifier(e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; OhrPharmaceutical); vitamin and mineral supplements (e.g., those describedin the Age-Related Eye Disease Study 1 (AREDS1; zinc and/orantioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein,zeaxanthin, and/or omega-3 fatty acids)); a cell-based therapy, forexample, NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC celltransplantation (StemCells), CNTO-2476 (Janssen). OpRegen (Cell CureNeurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics);a tissue factor antagonist (e.g., hI-con1; Iconic Therapeutics); analpha-adrenergic receptor agonist (e.g, brimonidine tartrate); a peptidevaccine (e.g., S-646240: Shionogi); an amyloid beta antagonist (e.g., ananti-beta amyloid monoclonal antibody, e.g., GSK-933776); an S1Pantagonist (e.g., an anti-SIP antibody, e.g., iSONEP™; Lpath Inc); aROBO4 antagonist (e.g., an anti-ROBO4 antibody, e.g., DS-7080a; DaiichiSankyo); a lentiviral vector expressing endostatin and angiostatin(e.g., RetinoStat); and any combination thereof. In some instances, AMDtherapeutic agents (including any of the preceding AMD therapeuticagents) can be co-formulated. For example, the anti-PDGFR antibodyREGN2176-3 can be co-formulated with aflibercept (EYLEA®). In someinstances, such a co-formulation can be administered in combination withan antibody of the invention. In some instances, the ocular disorder isAMD (e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith LUCENTIS® (ranibizumab) for treatment of an ocular disorder (e.g.,AMD, DME, DR, or RVO). LUCENTIS® (ranibizumab) may be administered, forexample, at 0.3 mg/eye or 0.5 mg/eye by intravitreal injection, forexample, every month. In some instances, the ocular disorder is AMD(e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith EYLEA® (aflibercept) for treatment of an ocular disorder (e.g.,AMD, DME, DR, or RVO). EYLEA® (aflibercept) may be administered, forexample, at 2 mg/eye by intravitreal injection, for example, every fourweeks (Q4W), or Q4W for the first three months, followed by injectionsonce every two months for maintenance. In some instances, the oculardisorder is AMD (e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith MACUGEN® (pegaptanib sodium) for treatment of an ocular disorder(e.g., AMD, DME, DR, or RVO). MACUGEN® (pegaptanib sodium) may beadministered, for example, at 0.3 mg/eye by intravitreal injection everysix weeks. In some instances, the ocular disorder is AMD (e.g., wetAMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith VISUDYNE® (verteporfin) in combination with photodynamic therapyfor treatment of an ocular disorder (e.g., AMD, DME, DR, or RVO).VISUDYNE® can be administered, for example, by intravenous infusion atany suitable dose (e.g., 6 mg/m² of body surface area) and deliveredonce every three months (e.g., over 10 minutes of infusion). In someinstances, the ocular disorder is AMD (e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith a PDGF antagonist for treatment of an ocular disorder (e.g., AMD,DME, DR, or RVO). Exemplary PDGF antagonists which may be used incombination with an antibody of the invention include an anti-PDGFantibody, an anti-PDGFR antibody, a small molecule inhibitor (e.g.,squalamine), an anti-PDGF-B pegylated aptamer such as FOVISTA® (E10030;Ophthotech/Novartis), or a dual PDGF/VEGF antagonist (e.g., a smallmolecule inhibitor (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or abispecific anti-PDGF/anti-VEGF antibody). For example, FOVISTA® can beadministered as an adjunct therapy to an antibody of the invention.FOVISTA® can be administered at any suitable dose, for example, from 0.1mg/eye to 2.5 mg/eye, e.g., at 0.3 mg/eye or 1.5 mg/eye, for example, byintravitreal injection, for example every four weeks (Q4W). OHR-102(squalamine lactate ophalmic solution, 0.2%) can be administered by eyedrop, for example, twice daily. OHR-102 can be administered incombination with VEGF antagonists such as LUCENTIS® or EYLEA®. In someembodiments, an antibody of the invention can be administered incombination with OHR-102, LUCENTIS®, and/or EYLEA®. In some instances,the ocular disorder is AMD (e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith RTH-258 for treatment of an ocular disorder (e.g., AMD, DME, DR, orRVO). RTH-258 can be administered, for example, by intravitrealinjection or eye infusion. For intravitreal injection, RTH-258 can beadministered at any suitable dose (e.g., 3 mg/eye or 6 mg/eye), forexample, once every four weeks (Q4W) for the first three months asloading, followed by injection every 12 weeks (Q12W) or every eightweeks (Q8W) for maintenance. In some instances, the ocular disorder isAMD (e.g., wet AMD).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith abicipar pegol for treatment of an ocular disorder (e.g., AMD, DME,DR, or RVO). Abicipar pegol can be administered, for example, byintravitreal injection. Abicipar pegol can be administered at anysuitable dose (e.g., 1 mg/eye, 2 mg/eye, 3 mg/eye, 4 mg/eye, or 4.2mg/eye), for example, once every four weeks (Q4W) for the first threemonths as loading, followed by injection every 12 weeks (Q12W) or everyeight weeks (Q8W) for maintenance. In some instances, the oculardisorder is AMD (e.g., wet AMD).

Any suitable DME and/or DR therapeutic agent can be administered incombination with an antibody of the invention, or an antibody conjugate,fusion protein, and/or polymeric formulation thereof, for treatment ofan ocular disorder (e.g., AMD, DME, DR, or RVO), including, but notlimited, to a VEGF antagonist (e.g., LUCENTIS® or EYLEA®), acorticosteroid (e.g., a corticosteroid implant (e.g., OZURDEX®(dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonideintravitreal implant)) or a corticosteroid formulated for administrationby intravitreal injection (e.g., triamcinolone acetonide)), orcombinations thereof. In some instances, the ocular disorder is DMEand/or DR.

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith LUCENTIS® (ranibizumab) for treatment of DME and/or DR (e.g., NPDRor PDR). LUCENTISS (ranibizumab) may be administered, for example, at0.3 mg/eye or 0.5 mg/eye by intravitreal injection, for example, everyfour weeks (Q4W).

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith EYLEA® (aflibercept) for treatment of DME and/or DR (e.g., NPDR orPDR). EYLEA® (aflibercept) may be administered, for example, at 2 mg/eyeby intravitreal injection, for example, every four weeks (Q4W), or Q4Wfor the first five months, followed by injections once every eight weeks(Q8W) for maintenance.

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith OZURDEX® (dexamethasone intravitreal implant) for treatment of DMEand/or DR. OZURDEX® can be administered as a 0.7 mg dexamethasoneintravitreal implant, which can be administered up to every six months.

An antibody of the invention, or an antibody conjugate, fusion protein,and/or polymeric formulation thereof, can be administered in combinationwith ILUVIEN® (dexamethasone intravitreal implant) for treatment of DMEand/or DR. OZURDEX® can be administered as a 0.19 mg fluocinoloneacetonide intravitreal implant, which can be eluted at a rate of 0.25μg/day, and can last up to about 36 months.

In some cases, the TAO/PRN treatment regimen or TAE treatment regimenmay be used to administer an AMD therapeutic agent (e.g., ranibizumab oraflibercept) in combination with an antibody of the invention, or anantibody conjugate, fusion protein, and/or polymeric formulationthereof. For the TAO/PRN regimen, following initial intravitrealinjections every four weeks (Q4W) (typically for about 3 months), thesubject is monitored monthly or every other month (or at even longerintervals), with injections administered in the event of evidence ofdisease activity (e.g., a decline in visual acuity or fluid on opticalcoherence tomography (OCT)). For the TAE regimen, a subject may betreated every four weeks (Q4W), followed by extending the interval oftreatment by a fixed number of weeks (e.g., +2 weeks) for eachsubsequent visit up to a maximal interval (e.g., every 6 weeks, ever 8weeks, every 10 weeks, or every 12 weeks). The eye(s) may be observedand treated at each visit, even if there is no evidence of diseaseactivity. If the macula appears wet (e.g., by OCT), the interval forinjections can be shortened (e.g., −2 weeks) until the macula appearsdry again. In some instances, the ocular disorder is AMD (e.g., wetAMD).

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-VEGF antibody, antibody conjugate, fusion protein, or polymericformulation and administration of an additional therapeutic agent occurwithin about one, two, three, four, or five months, or within about one,two or three weeks, or within about one, two, three, four, five, or sixdays, of each other. Antibodies, antibody conjugates, fusion proteins,and polymeric formulations of the invention can also be used incombination with radiation therapy.

An antibody, antibody conjugate, fusion protein, or polymericformulation of the invention (and any additional therapeutic agent) forprevention or treatment of an ocular disease or condition can beadministered by any suitable means, including but not limited to, forexample, ocular, intraocular, and/or intravitreal injection, and/orjuxtascleral injection, and/or subtenon injection, and/or superchoroidalinjection, and/or topical administration in the form of eye drops and/orointment. Such antibodies, antibody conjugates, fusion proteins, orpolymeric formulations of the invention may be delivered by a variety ofmethods, for example, intravitreally as a device and/or a depot thatallows for slow release of the compound into the vitreous, includingthose described in references such as Intraocular Drug Delivery, Jaffe,Jaffe, Ashton, and Pearson, editors. Taylor & Francis (March 2006). Inone example, a device may be in the form of a mini pump and/or a matrixand/or a passive diffusion system and/or encapsulated cells that releasethe compound for a prolonged period of time (Intraocular Drug Delivery,Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March2006). Additional approaches which may be used are described in SectionK below.

Formulations for ocular, intraocular, or intravitreal administration canbe prepared by methods and using excipients known in the art. Animportant feature for efficient treatment is proper penetration throughthe eye. Unlike diseases of the front of the eye, where drugs can bedelivered topically, retinal diseases typically benefit from a moresite-specific approach. Eye drops and ointments rarely penetrate theback of the eye, and the blood-ocular barrier hinders penetration ofsystemically administered drugs into ocular tissue. Accordingly, amethod of choice for drug delivery to treat retinal disease, such as AMDand CNV, is typically direct intravitreal injection. Intravitrealinjections are usually repeated at intervals which depend on thepatient's condition, and the properties and half-life of the drugdelivered. Additional approaches which may be used are described inSection K below.

The amount of antibody, antibody variant thereof, antibody conjugate,fusion protein, or polymeric formulation thereof which will be effectivein the treatment of a particular ocular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. Where possible, it is desirable todetermine the dose-response curve and the pharmaceutical compositions ofthe invention first in vitro, and then in useful animal model systemsprior to testing in humans.

Additional suitable administration means include parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, forexample, by injections, such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein. In some instances, an anti-VEGFantibody, antibody conjugate, fusion protein, or polymeric formulationof the invention may be administered intravenously, intramuscularly,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, topically, transdermally, byinhalation, by injection, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions

For the prevention or treatment of disease, the appropriate dosage of anantibody, antibody conjugate, fusion protein, or polymeric formulationof the invention (when used alone or in combination with one or moreother additional therapeutic agents) will depend on the type of diseaseto be treated, the type of antibody, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody, antibody conjugate, fusion protein, orpolymeric formulation is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg, 0.2 mg/kg,0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of antibody canbe an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. In some embodiments, the antibody used is about0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg. Onetypical daily dosage might range from about 1 μg/kg to 100 mg/kg ormore, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-VEGF antibody.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an antibody conjugate, fusion protein,or polymeric formulation of the invention (e.g., any described herein,e.g., in Section K below) in place of or in addition to an anti-VEGFantibody.

I. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of, or in additionto, an anti-VEGF antibody.

It is understood that any of the above articles of manufacture mayinclude an antibody conjugate, fusion protein, or polymeric formulationof the invention in place of, or in addition to, an anti-VEGF antibody.

J. Methods of Identifying Antibody Variants and Improving Antibodies

The invention provides methods of improving antibodies and identifyingantibody variants. In some instances, the methods involve identifyingone or more amino acid residue alterations that confers enhanced bindingof an antibody to a target molecule. In some instances, the methodsinvolve identifying one or more amino acid residue alterations thatconfers enhanced stability (e.g., thermal stability), functionalexpression, and/or protein folding of an antibody. In some instances,the invention provides methods of improving the binding affinity of anantibody to a target molecule. In some instances, the invention providesmethods of improving the stability (e.g., thermal stability), functionalexpression, and/or protein folding of an antibody. In some instances,the invention provides methods of improving the binding affinity of anantibody to a target molecule and improving the stability (e.g., thermalstability) of the antibody.

For example, the invention provides methods of identifying an amino acidresidue alteration that confers enhanced binding of an antibody to atarget molecule that involve one or more (e.g., 1, 2, or 3) of thefollowing steps: (a) providing a display library comprising nucleicacids encoding candidate antibody variants, wherein each candidateantibody variant comprises an amino acid residue alteration in the VH orthe VL compared to a reference antibody, and wherein amino acid residuealterations at every position of the VH or VL are present in the displaylibrary; (b) sorting the display library based on binding of thecandidate antibody variants to the target molecule to form a sortedlibrary, wherein the sorted library comprises candidate antibodyvariants with enhanced binding to the target molecule compared to thereference antibody; and (c) comparing the frequency at which each aminoacid residue alteration is present in the display library and in thesorted library as determined by massively parallel sequencing, therebydetermining whether each amino acid residue alteration is enriched inthe sorted library compared to the display library, whereby the aminoacid residue alteration is identified as conferring enhanced binding tothe target molecule if it is enriched in the sorted library compared tothe display library. In some instances, the method further includesidentifying an amino acid residue that confers enhanced stability to theantibody, for example, as described below.

In another example, the the invention provides methods of identifying anamino acid residue alteration that confers enhanced stability to anantibody that involve one or more (e.g., 1, 2, or 3) of the followingsteps: (a) providing a display library comprising nucleic acids encodingcandidate antibody variants, wherein each candidate antibody variantcomprises an amino acid residue alteration in the VH or the VL comparedto a reference antibody, and wherein amino acid residue alterations atevery position of the VH or VL are present in the display library; (b)sorting the display library based on binding of the candidate antibodyvariants to the target molecule to form a sorted library, wherein thesorted library comprises candidate antibody variants with enhancedstability compared to the reference antibody; and (c) comparing thefrequency at which each amino acid residue alteration is present in thedisplay library and in the sorted library as determined by massivelyparallel sequencing, thereby determining whether each amino acid residuealteration is enriched in the sorted library compared to the displaylibrary, whereby the amino acid residue alteration is identified asconferring enhanced stability to the antibody if it is enriched in thesorted library compared to the display library. In some instances, themethod further includes identifying an amino acid residue that confersenhanced binding of an antibody to a target molecule, for example, asdescribed above.

Any of the preceding methods may further include determining thefrequency at which each amino acid alteration is present in the displaylibrary and the sorted library by massively parallel sequencingfollowing step (b).

In some instances, an amino acid residue alteration may be enriched atleast 2-fold in the sorted library compared to the display library. Forexample, an amino acid residue alteration may be enriched 1.25-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 14-fold, 16-fold, or more in thesorted library compared to the display library.

A display library may include any suitable number of antibody variants,for example, from about 1×10³ to about 1×10¹² or more (e.g., about1×10³, about 1×10⁴, about 1×10⁵, about 1×10⁶, about 1×10⁷, about1.5×10⁷, about 2.5×10⁷, about 1×10⁸, about 5×10⁸, about 1×10⁹, about5×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹² or more) antibodyvariants. In some embodiments, the display library may include about3×10⁹ antibody variants. In other embodiments, the display library mayinclude about 8×10⁸ antibody variants.

In any of the preceding methods, an amino acid residue alteration may beencoded by any suitable codon set. For example, in some instances, theamino acid residue alteration is encoded by a degenerate codon set.Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. See also U.S. Pat. No. 7,985,840, which is incorporated hereinby reference in its entirety. For example, libraries as described aboveor in the Examples section below can be created by amino acidsubstitution with variant amino acids using the Kunkel method. See, forexample, Kunkel et al., Methods Enzymol. 154:367-382, 1987.

An amino acid residue alteration may be encoded by any suitable codonset. A codon set is a set of different nucleotide triplet sequences usedto encode desired variant amino acids. Codon sets can be representedusing symbols to designate particular nucleotides or equimolar mixturesof nucleotides as shown in below according to the IUB code.

IUB Codes

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T)

N (A or C or G or T)

As an illustrative example, in the codon set DVK, D can be nucleotides Aor G or T; V can be A or G or C; and K can be G or T. This codon set canpresent 18 different codons and can encode amino acids Ala, Trp, Tyr,Lys, Thr, Asn, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems. FosterCity. Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In any of the preceding methods, a degenerate codon set may be used toencode amino acid residue alterations. In some instances, the degeneratecodon set is an NNK or an NNS codon set, wherein N is A, C, G, or T; Kis G or T; and S is C or G. In particular instances, the degeneratecodon set is an NNK codon set.

It is to be understood that any suitable display approach known in theart or described herein may be used in conjunction with any of thepreceding methods. For example, the methods may involve phage display,bacterial display, yeast display, mammalian display, ribosome display,and/or mRNA display. In any of the preceding methods, any suitabledisplay library may be used. For example, the display library may beselected from the group consisting of a phage display library, abacterial display library, a yeast display library, a mammalian displaylibrary, a ribosome display library, and an mRNA display library. Inparticular embodiments, the display library is a phage display library.

Fusion polypeptides of an antibody variable domain can be displayed onthe surface of a cell, virus, phagemid, or other particle in a varietyof formats. These formats include, for example, scFv, Fab, andmultivalent forms of these fragments. The multivalent forms may be adimer of ScFv, Fab, or Fab′, herein referred to as (ScFv)₂, Fab₂ andF(ab)₂, respectively. Methods for displaying fusion polypeptidescomprising antibody fragments, on the surface of bacteriophage, are wellknown in the art, for example as described in patent publication numberWO 92/01047 and herein. Other patent publications, for example, WO92/20791; WO 93/06213; WO 93/11236, and WO 93/19172, describe relatedmethods. Other publications have shown the identification of antibodieswith artificially rearranged V gene repertoires against a variety ofantigens displayed on the surface of phage (see, e.g., Hoogenboom etal., J. Mol. Biol. 227: 381-388, 1992; and as disclosed in WO 93/06213and WO 93/11236).

In any of the preceding methods, the display library may be sorted(selected) and/or screened to identify, for example, high-affinitybinders to an antigen. Sorting may be performed as described herein orby other approaches known in the art. See, for example, U.S. Pat. No.7,985,840. In some embodiments, sorting may involve contacting thedisplay library with an immobilized antigen (e.g., target molecule orepitope thereof). In other embodiments, sorting may involve contactingthe display library with a soluble antigen. Antibody variants that havebeen selected can be further screened to characterize the antibodyvariant in terms of binding affinity (e.g., by SPR), stability, folding,structure (e.g., by X-ray crystallography), or other attributes.

Any of the preceding methods may involve massively parallel sequencing,for example, to determine the frequency that an amino acid residuealteration appears in a library following sorting (referred to as asorted library) as compared to the the frequency that the amino acidresidue alteration appears in an unsorted library. A wide variety ofapproaches for massively parallel sequencing are known in the art, andany suitable approach may be used in the methods of the invention. See,for example, Metzker, Nature Reviews Genetics 11: 31-36, 2010, which isincorporated by reference herein in its entirety. Exemplary approachesinclude massively parallel signature sequencing (MPSS), polonysequencing, pyrosequencing (454/Roche Diagnostics), ion semiconductorsequencing, single-molecule real-time sequencing, sequencing bysynthesis, sequencing by ligation. Commercially-available massivelyparallel sequencing platforms are available from Roche Diagnostics andother companies. The sequencing may be deep sequencing, ultra-deepsequencing, and/or next-generation sequencing.

In any of the preceding methods, the method may involve determining thesequence of at least about 100,000 reads or more (e.g., 100,000 reads;200,000 reads; 300,000 reads; 400,000 reads; 500,000 reads; 600,000reads; 700,000 reads; 800,000 reads; 900,000 reads; 1,000,000 reads;2×10⁶ reads; 3×10⁶ reads; 4×10⁶ reads; 5×10⁶ reads; 6×10⁶ reads; 7×10⁶reads; 8×10⁶ reads; 9×10⁶ reads; 10⁷ reads; 10⁸ reads; 10⁹ reads; or10¹⁰ reads or more). The method may involve sequencing at any suitabledepth.

In any of the preceding methods, the antibody may be a monoclonalantibody. In any of the preceding methods, the antibody may be an IgGantibody. In any of the preceding methods, the antibody may be anantibody fragment. The antibody fragment may be selected from the groupconsisting of Fab, scFv, Fv, Fab′, Fab-C, Fab′-SH, F(ab′)₂, and diabody.In particular instances, the antibody fragment is a Fab.

In any of the preceding methods, the dual-specific antibody may be amonoclonal antibody. In any of the preceding methods, the dual-specificantibody may be an IgG antibody. In any of the preceding methods, thedual-specific antibody may be an antibody fragment. The antibodyfragment may be selected from the group consisting of Fab, scFv, Fv,Fab-C, Fab′, Fab′-SH, F(ab′)₂, and diabody. In particular instances, theantibody fragment is a Fab.

Any of the preceding methods may further involve generating an antibodythat has been identified by the steps of the method. The methodsdescribed above may be used with any of the antibodies described herein.

K. Ocular Long-Acting Delivery Approaches for Anti-VEGF Antibodies

The invention provides compositions for treatment of ocular disorders,which may be used for long-acting delivery of anti-VEGF antibodies(including any anti-VEGF antibody described herein, such as G6.31 AARR)to the eye. For example, the invention provides antibody conjugates thatinclude an anti-VEGF antibody described herein (e.g., Fab or Fab-Cantibody conjugates). The invention also provides fusion proteins (e.g.,Fab fusion proteins). In other aspects, the invention providesformulations (e.g., polymeric formulations) that include an anti-VEGFantibody described herein. The invention also provides devices that canbe used for ocular administration of an anti-VEGF antibody describedherein. The invention further provides pharmaceutical compositions thatinclude antibody conjugates, fusion proteins, and/or formulations (e.g.,polymeric formulations) described herein. These compositions can be usedin any of the therapeutic methods described herein, for example, methodsof treating an ocular disorder (e.g., AMD (e.g., wet AMD), DME, DR(e.g., NPDR or PDR), or RVO (e.g., CRVO or BRVO)).

1. Antibody Conjugates

The invention provides antibody conjugates that include an anti-VEGFantibody and a polymer covalently attached to the antibody. Theanti-VEGF antibody may be covalently attached to the polymer in anirreversible fashion or a reversible fashion. Any suitable polymer maybe used, including those described herein or others known in the art.The polymer may be a hydrophilic polymer or a hydrophobic polymer. It isto be understood that a hydrophilic polymer may be a water-solublepolymer. Any suitable hydrophilic polymer may be used, for example, ahydrophilic polymer described in International Patent ApplicationPublication No. WO 2011/066417 and/or Pelegri-O'Day et al. J. Am. Chem.Soc. 136:14323-14332, 2014, which are incorporated herein by referencein their entirety. Exemplary, non-limiting hydrophilic polymers that canbe used include hyaluronic acid (HA), polyethylene glycol (PEG; alsoknown as poly(ethylene glycol)) (e.g., straight-chain PEG, branched PEG,comb-like PEG, and dendritic PEG), poly[ethylene oxide)-co-(methyleneethylene oxide)], poly(poly(ethylene glycol) methyl ether methacrylate)(pPEGMA), agarose, alginate, carageenans, carboxymethylcellulose,cellulose, cellulose derivatives, chitosan, chondroitin sulfate,collagen, dermatan sulfate, dextran, dextran sulfate, fibrin,fibrinogen, fibronectin, fucoidan, gelatin, glycosaminoglycans (GAGs), aglycopolymer, heparin, heparin sulfate, a highly-branched polysaccharide(e.g., a galactose dendrimer), keratan sulfate, methyl cellulose,hydroxypropylmethylcellulose (HPMC),poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), pectins, pectinderivatives, pentosane polysulfate, starch, hydroxylethyl starch (HES),styrene, vitronectin, poly(acrylic acid), poly(methacrylic acid),poly(acrylamide), poly(acrylic acid), poly(amines), poly(amino acids),poly(carboxybetaine) (PCB), polyelectrolytes, poly(glutamic acid) (PGA),poly(glycerol) (PG) (e.g., linear, midfunctional, hyperbranched, orlinear hyperbranched PG), poly(maleic acid), poly(2-oxazoline) (POZ),poly(2-ethyl-2-oxazoline, polysialic acid (PSA), polystyrene,polystyrene derivatives (e.g., charged polystyrene derivatives),poly(styrenesulfonate-co-PEGMA), polyvinylpyrrolidone (PVP),poly(N-acryloylmorpholine) (pNAcM), and copolymers thereof. In someinstances, the polymer is a hydrophobic polymer, for example,poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), andpolyglycolide (PGA). The polymer may be biodegradable and/orbiocompatible.

By way of example, the polymer may include any suitable number ofmonomers, for example, between 2 and about 1×10⁴ monomers (e.g., about10, about 50, 100, about 200, about 300, about 400, about 500, about600, about 700, about 800, about 900, about 1000, about 2000, about3000, about 4000, about 5000, about 6000, about 7000, about 8000, about9000, or about 1×10⁴ monomers), or more. For example, the polymer mayinclude between about 50 and about 250 monomers, about 50 and about 500monomers, between about 50 and about 1000 monomers, between about 50 andabout 2000 monomers, between about 50 and about 3000 monomers, betweenabout 50 and about 4000 monomers, between about 50 and about 5000monomers, between about 50 and about 6000 monomers, between about 50 andabout 7000 monomers, between about 50 and about 8000 monomers, betweenabout 50 and about 9000 monomers, between about 50 and about 10000monomers, between about 100 and about 250 monomers, about 100 and about500 monomers, between about 100 and about 1000 monomers, between about100 and about 2000 monomers, between about 100 and about 3000 monomers,between about 100 and about 4000 monomers, between about 100 and about5000 monomers, between about 100 and about 6000 monomers, between about100 and about 7000 monomers, between about 100 and about 8000 monomers,between about 100 and about 9000 monomers, between about 100 and about10000 monomers, between about 250 and about 500 monomers, between about250 and about 1000 monomers, between about 250 and about 2000 monomers,between about 250 and about 3000 monomers, between about 250 and about4000 monomers, between about 250 and about 5000 monomers, between about250 and about 6000 monomers, between about 250 and about 7000 monomers,between about 250 and about 8000 monomers, between about 250 and about9000 monomers, between about 250 and about 10000 monomers. between about500 and about 1000 monomers, between about 500 and about 2000 monomers,between about 500 and about 3000 monomers, between about 500 and about4000 monomers, between about 500 and about 5000 monomers, between about500 and about 6000 monomers, between about 500 and about 7000 monomers,between about 500 and about 8000 monomers, between about 500 and about9000 monomers, or between about 500 and about 10000 monomers. In someinstances, the polymer may include about 500 monomers.

The invention provides an antibody conjugate that includes an anti-VEGFantibody (e.g., an anti-VEGF antibody described herein, such as G6.31AARR) covalently attached to a HA polymer. Such antibody conjugates aresometimes referred to herein as “HA conjugates.” In some instances, theHA polymer has a molecular weight of about 2.5 megadalton (MDa) or lower(e.g., about 2.5 MDa or lower, about 2.4 MDa or lower, about 2.3 MDa orlower, about 2.2. MDa or lower, about 2.1 MDa or lower, about 2.0 MDa orlower, about 1.9 MDa or lower, about 1.8 MDa or lower, about 1.7 MDa orlower, about 1.6 MDa or lower, about 1.5 MDa or lower, about 1.4 MDa orlower, about 1.3 MDa or lower, about 1.2 MDa or lower, about 1.1 MDa orlower, about 1.0 MDa or lower, about 900 kDa or lower, about 800 kDa orlower, about 700 kDa or lower, about 600 kDa or lower, about 500 kDa orlower, about 400 kDa or lower, about 300 kDa or lower, about 200 kDa orlower, or about 100 kDa or lower). In some instances, the HA polymer hasa molecular weight of about 1 MDa or lower (e.g., about 1.0 MDa orlower, about 900 kDa or lower, about 800 kDa or lower, about 700 kDa orlower, about 600 kDa or lower, about 500 kDa or lower, about 400 kDa orlower, about 300 kDa or lower, about 200 kDa or lower, or about 100 kDaor lower). In some instances, the HA polymer has a molecular weightbetween about 25 kDa and about 2.5 MDa (e.g., between about 25 kDa andabout 2.5 mDa, between about 25 kDa and about 2 MDa, between about 25kDa and about 1.5 MDa, between about 25 kDa and about 1 MDa, betweenabout 25 kDa and about 900 kDa, between about 25 kDa and about 800 kDa,between about 25 kDa and about 700 kDa, between about 25 kDa and about600 kDa, between about 25 kDa and about 500 kDa, between about 100 kDaand about 2.5 mDa, between about 100 kDa and about 2 MDa, between about100 kDa and about 1.5 MDa, between about 100 kDa and about 1 MDa,between about 100 kDa and about 900 kDa, between about 100 kDa and about800 kDa, between about 100 kDa and about 700 kDa, between about 100 kDaand about 600 kDa, between about 100 kDa and about 500 kDa, betweenabout 250 kDa and about 2.5 MDa, between about 250 kDa and about 2 MDa,between about 250 kDa and about 1.5 MDa, between about 250 kDa and about1 MDa, between about 250 kDa and about 900 kDa, between about 250 kDaand about 800 kDa, between about 250 kDa and about 700 kDa, betweenabout 250 kDa and about 600 kDa, between about 250 kDa and about 500kDa, between about 500 kDa and about 2.5 MDa, between about 500 kDa andabout 2 MDa, between about 500 kDa and about 1.5 MDa, between about 500kDa and about 1 MDa, between about 500 kDa and about 900 kDa, betweenabout 500 kDa and about 800 kDa, between about 500 kDa and about 700kDa, between about 500 kDa and about 600 kDa, between about 1 MDa andabout 2.5 MDa, between about 1 MDa and about 2 MDa, between about 1 MDaand about 1.5 MDa, between about 1 MDa and about 1.25 MDa, between about1.25 MDa and about 2.5 MDa, between about 1.25 MDa and about 2 MDa,between about 1.25 MDa and about 1.5 MDa, between about 1.5 MDa andabout 2.5 MDa, between about 1.5 MDa and about 2 MDa, between about 1.5MDa and about 1.75 MDa, or between 1.75 MDa and about 2.5 MDa).

In some instances, the HA polymer has a molecular weight between about25 kDa and about 500 kDa (e.g., between about 25 kDa and about 500 kDa,between about 25 kDa and about 450 kDa, between about 25 kDa and about400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDaand about 300 kDa, between about 25 kDa and about 300 kDa, between about25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa,between about 25 kDa and about 150 kDa, between about 25 kDa and about100 kDa, between about 25 kDa and about 50 kDa, between about 40 kDa andabout 500 kDa, between about 40 kDa and about 450 kDa, between about 40kDa and about 400 kDa, between about 40 kDa and about 350 kDa, betweenabout 40 kDa and about 300 kDa, between about 40 kDa and about 300 kDa,between about 40 kDa and about 250 kDa, between about 40 kDa and about200 kDa, between about 40 kDa and about 150 kDa, between about 40 kDaand about 100 kDa, between about 40 kDa and about 50 kDa, between about50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa,between about 50 kDa and about 400 kDa, between about 50 kDa and about350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDaand about 300 kDa, between about 50 kDa and about 250 kDa, between about50 kDa and about 200 kDa, between about 50 kDa and about 150 kDa,between about 50 kDa and about 100 kDa, between about 50 kDa and about75 kDa, between about 100 kDa and about 500 kDa, between about 100 kDaand about 450 kDa, between about 100 kDa and about 400 kDa, betweenabout 100 kDa and about 350 kDa, between about 100 kDa and about 300kDa, between about 100 kDa and about 300 kDa, between about 100 kDa andabout 250 kDa, between about 100 kDa and about 200 kDa, between about100 kDa and about 150 kDa, between about 150 kDa and about 500 kDa,between about 150 kDa and about 450 kDa, between about 150 kDa and about400 kDa, between about 150 kDa and about 350 kDa, between about 150 kDaand about 300 kDa, between about 150 kDa and about 300 kDa, betweenabout 150 kDa and about 250 kDa, between about 150 kDa and about 200kDa, between about 175 kDa and about 500 kDa, between about 175 kDa andabout 450 kDa, between about 175 kDa and about 400 kDa, between about175 kDa and about 350 kDa, between about 175 kDa and about 300 kDa,between about 175 kDa and about 300 kDa, between 175 200 kDa and about250 kDa, between about 175 kDa and about 225 kDa, between about 200 kDaand about 500 kDa, between about 200 kDa and about 450 kDa, betweenabout 200 kDa and about 400 kDa, between about 200 kDa and about 350kDa, between about 200 kDa and about 300 kDa, between about 200 kDa andabout 300 kDa, between about 200 kDa and about 250 kDa, or between about200 kDa and about 225 kDa).

In some instances, the HA polymer has a molecular weight between about100 kDa and about 250 kDa (e.g., about 100 kDa, about 110 kDa, about 120kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa,about 220 kDa, about 230 kDa, about 240 kDa, or about 250 kDa). Inparticular instances, the HA polymer has a molecular weight of about 200kDa.

Any of the preceding molecular weights may be a weight-average molecularweight (also known as weight-average molar mass).

In some instances, any of the preceding HA polymers is linear, i.e., notcross-linked.

In other instances, the invention provides an antibody conjugate thatincludes an anti-VEGF antibody (e.g., an anti-VEGF antibody describedherein) covalently attached to a PEG polymer. Such antibody conjugatesare sometimes referred to as “PEG conjugates” herein. Any suitable PEGpolymer may be used. The PEG may be a branched PEG, a star PEG, or acomb PEG. The PEG polymer may be, for example, a PEG tetramer, a PEGhexamer, or a PEG octamer. In some instances, the antibody conjugateincludes an anti-VEGF antibody (e.g., an anti-VEGF antibody describedherein, such as G6.31 AARR) covalently attached to a PEG dendrimer. PEGpolymers are commercially available, for example, from JenKemTechnology, Quanta BioDesign, NOF America Corporation, and othervendors.

In some instances, the PEG polymer has a molecular weight between about1 kDa and about 500 kDa (e.g., between about 1 kDa and about 500 kDa,between about 1 kDa and about 450 kDa, between about 1 kDa and about 400kDa, between about 1 kDa and about 350 kDa, between about 1 kDa andabout 300 kDa, between about 1 kDa and about 300 kDa, between about 1kDa and about 250 kDa, between about 1 kDa and about 200 kDa, betweenabout 1 kDa and about 150 kDa, between about 1 kDa and about 100 kDa,between about 1 kDa and about 50 kDa, between about 10 kDa and about 500kDa, between about 10 kDa and about 450 kDa, between about 10 kDa andabout 400 kDa, between about 10 kDa and about 350 kDa, between about 10kDa and about 300 kDa, between about 10 kDa and about 300 kDa, betweenabout 10 kDa and about 250 kDa, between about 10 kDa and about 200 kDa,between about 10 kDa and about 150 kDa, between about 10 kDa and about100 kDa, between about 10 kDa and about 50 kDa, between about 20 kDa andabout 500 kDa, between about 20 kDa and about 450 kDa, between about 20kDa and about 400 kDa, between about 20 kDa and about 350 kDa, betweenabout 20 kDa and about 300 kDa, between about 20 kDa and about 300 kDa,between about 20 kDa and about 250 kDa, between about 20 kDa and about200 kDa, between about 20 kDa and about 150 kDa, between about 20 kDaand about 100 kDa, between about 20 kDa and about 75 kDa, between about30 kDa and about 500 kDa, between about 30 kDa and about 450 kDa,between about 30 kDa and about 400 kDa, between about 30 kDa and about350 kDa, between about 30 kDa and about 300 kDa, between about 30 kDaand about 300 kDa, between about 30 kDa and about 250 kDa, between about30 kDa and about 200 kDa, between about 30 kDa and about 150 kDa,between about 40 kDa and about 500 kDa, between about 40 kDa and about450 kDa, between about 40 kDa and about 400 kDa, between about 40 kDaand about 350 kDa, between about 40 kDa and about 300 kDa, between about40 kDa and about 300 kDa, between about 40 kDa and about 250 kDa,between about 40 kDa and about 200 kDa, between about 50 kDa and about500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDaand about 400 kDa, between about 50 kDa and about 350 kDa, between about50 kDa and about 300 kDa, between about 50 kDa and about 300 kDa,between 50 200 kDa and about 250 kDa, or between about 50 kDa and about225 kDa).

In some instances, the PEG polymer has a molecular weight between about5 kDa and about 250 kDa (e.g., about 1 kDa, about 5 kDa, about 10 kDa,about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa,about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa,about 90 kDa, 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa,about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about230 kDa, about 240 kDa, or about 250 kDa). In particular instances, thePEG polymer has a molecular weight of about 20 kDa. In other instances,the PEG polymer has a molecular weight of about 40 kDa.

Any of the preceding molecular weights may be a weight-average molecularweight (also known as weight-average molar mass).

In some instances, the PEG polymer is a PEG tetramer. PEG tetramers arecommercially available, for example, NOF America SUNBRIGHT® PTE-400MA,PTE-200MA, PTE-100MA, and JenKem Technology USA 4 arm PEG maleimide(Cat. No. 4ARM-MAL). In some instances, the PEG tetramer has apentaerythritol core. For example, in some instances, the PEG tetramerincludes a structure of formula (I), wherein n is independently anysuitable integer:

In another example, in some instances, the PEG polymer is a PEG hexamer.PEG hexamers are commercially available, for example, JenKem TechnologyUSA 6 arm PEG amine (Cat. No. 6ARM(DP)-NH2HCl), or PEG hexamers fromQuanta BioDesign. In some instances, the PEG hexamer includes adipentylerythritol core.

In some instances, the PEG polymer is a PEG octamer. PEG octamers arecommercially available, for example, NOF America SUNBRIGHT® HGEO seriesor JenKem Technology USA 8 arm PEG maleimide (Cat. No. 8ARM(TP)-MAL). Insome instances, the PEG octamer may include a tripentaerithritol core.For example, in some instances, the PEG octamer includes a structure offormula (II), wherein n is independently any suitable integer.

In yet another example, in some instances, the PEG octamer includes atripentaerythritol core.

It is to be understood that any suitable conjugation approach, includingthose described herein and others known in the art, may be used toconjugate an anti-VEGF antibody of the invention to a polymer. Forexample, the polymer may be conjugated to any suitable proteinfunctional group, including a primary amine group, a carboxyl group, asulfhydryl group, or a carbonyl group. Any suitable chemical reactivegroup may be used to target the protein functional group, for example,carbodiimide (e.g., EDC), NHS ester, imidoester, pentafluorophenylester, hydroxymethyl phosphine, maleimide, haloacetyl (e.g., bromoacetylor iodoacetyl), pyridyldisulfide, thiosulfonate, vinylsulfone,hydrazine, alkoxyamine, diazirine, aryl azide, isocyanate, or othersknown in the art. See, for example, Hermanson, Bioconjugate Techniques,3^(rd) Edition, 2013.

Any of the preceding antibody conjugates may have a hydrodynamic radiusbetween about 5 nm and about 200 nm (e.g., about 5 nm, about 10 nm,about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170nm, about 180 nm, about 190 nm, or about 200 nm). In some instances, theantibody conjugate has a hydrodynamic radius between about 5 nm andabout 150 nm (e.g., about 5 nm, about 10 nm, about 20 nm, about 30 nm,about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140nm, or about 150 nm). In some instances, the antibody conjugate has ahydrodynamic radius between about 5 nm and about 100 nm (e.g., about 5nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm,about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm). Insome instances, the antibody conjugate has a hydrodynamic radius betweenabout 5 nm and about 60 nm (e.g., about 5 nm, about 10 nm, about 20 nm,about 30 nm, about 40 nm, about 50 nm, or about 60 nm). In someinstances, the antibody conjugate has a hydrodynamic radius betweenabout 25 nm and about 35 nm (e.g., about 25 nm, about 26 nm, about 27nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm,about 33 nm, about 34 nm, or about 35 nm). In some instances, thehydrodynamic radius is about 28 nm.

In some instances, the antibody conjugate has a hydrodynamic radiusbetween about 10 nm and about 200 nm, between about 10 nm and about 180nm, between about 10 nm and about 160 nm, between about 10 nm and about140 nm, between about 10 nm and about 120 nm, between about 10 nm andabout 100 nm, between about 10 nm and about 80 nm, between about 10 nmand about 60 nm, between about 10 nm and about 50 nm, between about 10nm and about 40 nm, between about 10 nm and about 30 nm, between about20 nm and about 200 nm, between about 20 nm and about 180 nm, betweenabout 20 nm and about 160 nm, between about 20 nm and about 140 nm,between about 20 nm and about 120 nm, between about 20 nm and about 100nm, between about 20 nm and about 80 nm, between about 20 nm and about60 nm, between about 20 nm and about 50 nm, between about 20 nm andabout 40 nm, between about 20 nm and about 30 nm, between about 30 nmand about 200 nm, between about 30 nm and about 180 nm, between about 30nm and about 160 nm, between about 30 nm and about 140 nm, between about30 nm and about 120 nm, between about 30 nm and about 100 nm, betweenabout 30 nm and about 80 nm, between about 30 nm and about 60 nm,between about 30 nm and about 50 nm, between about 30 nm and about 40nm, between about 40 nm and about 200 nm, between about 40 nm and about180 nm, between about 40 nm and about 160 nm, between about 40 nm andabout 140 nm, between about 40 nm and about 120 nm, between about 40 nmand about 100 nm, between about 40 nm and about 80 nm, between about 40nm and about 60 nm, between about 40 nm and about 50 nm, between about50 nm and about 200 nm, between about 50 nm and about 180 nm, betweenabout 50 nm and about 160 nm, between about 50 nm and about 140 nm,between about 50 nm and about 120 nm, between about 50 nm and about 100nm, between about 50 nm and about 80 nm, between about 50 nm and about60 nm, between about 60 nm and about 200 nm, between about 60 nm andabout 180 nm, between about 60 nm and about 160 nm, between about 60 nmand about 140 nm, between about 60 nm and about 120 nm, between about 60nm and about 100 nm, or between about 60 nm and about 80 nm.

In some instances, the antibody conjugate is a prodrug antibodyconjugate (also referred to as a carrier-linked prodrug) in which ananti-VEGF antibody (e.g., an anti-VEGF antibody described herein, e.g.,G6.31 AARR) is reversibly conjugated to a carrier (e.g., a hydrogel),for example, via a linker (e.g., a reversible prodrug linker). Thisapproach is described further, for example, in International PatentApplication Publication Nos. WO 2006/003014, WO 2009/095479, WO2011/012715, WO 2013/053856, and WO 2014/056923, which are incorporatedherein by reference in their entirety. Such prodrug antibody conjugatesare commercially available from Ascendis Pharma (e.g., the TransContechnology platform). The linker may have inherent self-cleavingproperties (e.g., the linker may be non-enzymatically hydrolyzed uponadministration to the eye), leading to time-controlled release of theanti-VEGF antibody in the eye (e.g., vitreous).

For example, in some instances, the invention provides a prodrugantibody conjugate which includes an anti-VEGF antibody (e.g., ananti-VEGF antibody described herein, e.g., G6.31 AARR) that iscovalently attached to a carrier by a linker. In particular instances,the linker is a reversible prodrug linker. In some instances, thecarrier includes a polymer, for example, PEG. The PEG may be linear,branched, multi-arm, or dendritic PEG, for example. In some instances,the carrier is a hydrogel, including a biodegradable hydrogel. Anysuitable hydrogel can be used, for example, a PEG-based hydrogel. APEG-based hydrogel may include, for example, at least 10% PEG, at least20% PEG, at least 30% PEG, or more. The hydrogel may be in the shape ofmicroparticulate beads. Such microparticulate beads can have a diameterof about 1 μm to about 1000 μm, e.g., about 5 μm to about 500 μm, about10 μm to about 100 μm, about 20 μm to about 100 μm, or about 20 μm toabout 80 μm. Bead diameters can be measured when the microparticulatebeads are suspended in an isotonic aqueous buffer. In some instances,the hydrogel may be any hydrogel disclosed in WO 2006/003014 or WO2011/012715.

In any of the preceding antibody conjugates, the antibody may be anantibody fragment that binds VEGF, for example, an antibody fragment ofan anti-VEGF antibody described herein that binds VEGF. In someinstances, the antibody fragment is selected from the group consistingof Fab, Fab′, Fab-C, Fab′-SH, Fv, scFv, and (Fab)₂ fragments. Inparticular instances, the antibody fragment is an Fab, an Fab′, or anFab-C. In some instances, the antibody fragment is an Fab-C.

Any of the preceding antibody conjugates may have an ocular half-lifethat is increased relative to a reference antibody that is notcovalently attached to the polymer (e.g., the hydrophilic polymer). Insome instances, the ocular half-life is increased at least about 2-fold(e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, ormore) relative to the reference antibody. In some instances, the ocularhalf-life is increased at least about 4-fold relative to the referenceantibody. In some instances, the ocular half-life is a vitrealhalf-life. In some instances, the reference antibody is identical to theantibody of the antibody conjugate. In other cases, the referenceantibody is non-identical to the antibody of the antibody conjugate.

Any of the preceding antibody conjugates may have an ocular clearancethat is that is decreased relative to a reference antibody that is notcovalently attached to the polymer (e.g., the hydrophilic polymer). Insome instances, the clearance is decreased at least about 2-fold (e.g.,about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold,about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold,about 14-fold, about 16-fold, about 18-fold, about 20-fold, or more)relative to the reference antibody. In some instances, the clearance isdecreased at least about 4-fold relative to the reference antibody. Insome instances, the clearance is clearance from the vitreous. In someinstances, the reference antibody is identical to the antibody of theantibody conjugate. In other cases, the reference antibody isnon-identical to the antibody of the antibody conjugate.

In some instances, the time period between two intraocularadministrations (e.g., by intravitreal injection) of any of thepreceding antibody conjugates (e.g., HA conjugates, PEG conjugates, andprodrug antibody conjugates) is at least 1 month, e.g., at least 1month, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, atleast 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks,at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44weeks, at least 48 weeks, at least 52 weeks or more. In some cases, themaximum period between two intraocular administrations is no longer thenfour years, e.g., no longer than three years, no longer than two years,or no longer than one year. The antibody conjugate can be administered,for example, every two to twelve months, e.g., every four to ten months.In some instances, the antibody conjugate is administered every sixmonths.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the antibody conjugates describedabove (e.g., HA conjugates, PEG conjugates, and prodrug antibodyconjugates). In certain embodiments, the composition comprises one ormore additional compounds. In certain embodiments, the additionalcompound binds to a second biological molecule selected from the groupconsisting of IL-1β; IL-6; IL-6R; PDGF; angiopoietin; angiopoietin 2;Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk, such as complement pathway components C2,factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3;HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof. For example, in someinstances, the additional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

The invention further provides compositions (e.g., pharmaceuticalcompositions) that include any of the antibody conjugates describedabove (e.g., HA conjugates, PEG conjugates, and prodrug antibodyconjugates) and an additional VEGF antagonist.

2. Fusion Proteins

The invention provides fusion proteins that include an anti-VEGFantibody (e.g., any anti-VEGF antibody described herein, e.g., G6.31AARR) and an ocular binding domain. The ocular binding domain may bind,for example, to a biological substance found in the eye (e.g., thecornea, vitreous, retina, retina pigment epithelium, or choroid), whichmay increase ocular (e.g., vitreal) residence time (for example, byincreasing the half-life and/or decreasing clearance) of the antibody.The ocular binding domain may bind to any suitable biological substance,including, for example, an extracellular matrix component. For example,suitable biological substances in the eye (e.g., the vitreous) mayinclude an extracellular matrix component such as a carbohydrate (e.g.,a charged carbohydrate (e.g., a glycosaminoglycan)), a glycoprotein(e.g., fibrillin and opticin), and a protein (e.g., a collagen (e.g.,collagen types I-XXVII, particularly collagen II, collagen IX, collagenV, collagen VI, collagen XI, and heterotypic collagen fibrils thereof),or other extracellular matrix components described, for example, in LeGoff et al., Eye 22:1214-1222, 2008. In some instances, theextracellular matrix component is a glycosaminoglycan, for example,hyaluronic acid (HA) or a proteoglycan (e.g., chondroitin sulfate orheparin sulfate). In particular instances, the glycosaminoglycan is HA.HA binding domains, as well as fusion proteins that include HA bindingdomains, are described, for example, in Park et al., MolecularPharmaceutics 6(3):801-812, 2009; U.S. Pat. Nos. 5,986,052, 7,183,377,7,723,472, and 8,846,034; U.S. Patent Application Publication No.2004/005277; and International Patent Application Nos. WO 1998/052590,WO 2010/045506, WO 2014/099997, WO 2015/198243, WO 2015/110809, whichare incorporated herein by reference in their entirety. The ocularbinding domain may be covalently attached to the antibody, for example,by being recombinantly fused to the anti-VEGF antibody. In otherinstances, the ocular binding domain may be covalently conjugated ornon-covalently conjugated (e.g., by a biotin-strepavidin linkage) to theanti-VEGF antibody.

For example, the invention provides a fusion protein that includes ananti-VEGF antibody (e.g., any anti-VEGF antibody described herein, suchas G6.31 AARR) covalently attached to an HA binding domain. In someinstances, the HA binding domain is covalently attached to the heavychain or the light chain of the antibody. For example, the HA bindingdomain is covalently attached to the heavy chain. In another example,the HA binding domain is covalently attached to the light chain. The HAbinding domain can be covalently attached to the anti-VEGF antibody atany suitable site, for example, the N-terminus, the C-terminus, or aninternal site (e.g., an insertion). The HA binding domain can becovalently attached to the C-terminus of the heavy chain or to theC-terminus of the light chain. For example, in some instances, the HAbinding domain is covalently attached to the C-terminus of the heavychain. In other instances, the HA binding domain is covalently attachedto the C-terminus of the light chain.

In any of the preceding fusion proteins, the fusion protein may furtherinclude a linker, the linker being positioned between the antibody andthe HA binding domain. Any suitable linker may be used, for example, a(Gly_(n)-Ser_(n))_(n) or (Ser_(n)-Gly_(n))_(n) linker, where n isindependently an integer equal to or greater than 1 (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or more). WO 2014/099997 describes anumber of linkers, any of which may be used in the fusion proteins ofthe invention. In some instances, a linker may include the amino acidsequence of GGGGS (SEQ ID NO: 61). In some instances, a linker mayconsist of the amino acid sequence of GGGGS (SEQ ID NO: 61). Othersuitable linkers include (Gly₄Ser)₄, (Gly₄Ser)₃, and the like. In someinstances, serine can be replaced with alanine (e.g., (Gly₄Ala) or(Gly₃Ala)).

In any of the preceding fusion proteins, the antibody may be an antibodyfragment that binds VEGF. In some instances, the antibody fragment isselected from the group consisting of Fab, Fab′, Fab-C, Fab′-SH, Fv,scFv, and (Fab)₂ fragments. For example, in some instances, the antibodyfragment is an Fab, an Fab′, or an Fab-C. In some instances, the HAbinding domain is covalently attached to the C-terminus of the CH1domain of the Fab. In other instances, the HA binding protein iscovalently attached to the C-terminus of the CL domain of the Fab.

In any of the preceding fusion proteins, the HA binding domain may beselected from the group consisting of a link module, a G1 domain, alysine-rich oligopeptide, and other HA binding domains known in the art.For example, the HA binding domain may be any HA binding domaindescribed in Park et al., Molecular Pharmaceutics 6(3):801-812, 2009;U.S. Pat. Nos. 5,986,052, 7,183,377, 7,723,472, and 8.846,034; U.S.Patent Application Publication No. 2004/005277: and/or InternationalPatent Application Nos. WO 1998/052590, WO 2010/045506, WO 2014/099997,WO 2015/198243. WO 2015/110809. For example, the HA binding domain maybe HA10, HA10.1. HA10.2, HA11, or HA11.1, as described in WO2014/099997.

In some instances, the HA binding domain is a link module. Any suitablelink module may be used. For example, in some instances, the link moduleis selected from the group consisting of TSG6, CD44, lymphatic vesselendothelial hyaluronan receptor 1 (LYVE-1), hyaluronan and proteoglycanlink protein (HAPLN) 1, HAPLN2, HAPLN3, HAPLN4, aggrecan, brevican,neurocan, phosphacan, versican, CAB61358, KIA0527, stabilin-1, andstabilin-2 link modules, or variants thereof. In some instances, thelink module is a TSG6 link module, for example, a human TSG6 linkmodule. In some instances, the link module of the HA binding proteinTSG6 may include amino acid residues 36-128 of human TSG6 (UniProtAccession No. P98066).

Any of the preceding fusion proteins may further include at least oneadditional HA binding domain. For example, the fusion protein mayfurther include at least 1, 2, 3, 4, 5, 6, 7, or 8 additional HA bindingdomain(s). In some instances, the at least one additional HA bindingdomain is covalently attached to the heavy chain or the light chain ofthe antibody. For example, in some instances, the at least oneadditional HA binding domain is covalently attached to the heavy chainof the antibody. In other instances, the at least one additional HAbinding domain is covalently attached to the light chain of theantibody. In some instances, the at least one additional HA bindingprotein is linked to the antibody by a linker. Any suitable linker maybe used, such as a linker described above. In some instances, the linkerincludes the amino acid sequence of GGGGS (SEQ ID NO: 61). In someinstances, the linker consists of the amino acid sequence of GGGGS (SEQID NO: 61). In some instances, a first HA binding domain is covalentlyattached to the heavy chain, and a second HA binding domain iscovalently attached to the light chain. The at least one additional HAbinding domain can be covalently attached to the anti-VEGF antibody atany suitable site, for example, the N-terminus, the C-terminus, or aninternal site (e.g., an insertion). In instances where the antibodyincludes more than one additional HA binding domains in an antibody, theinsertions may be at a single site in the antibody or at multipledistinct sites in the antibody. For example, in some instances, thefirst HA binding domain is linked to the C-terminus of the heavy chainand the second HA binding domain is linked to the C-terminus of thelight chain. In some instances, the at least one additional HA bindingprotein is selected from the group consisting of a link module, a G1domain, and a lysine-rich oligopeptide. For example, in some instances,the at least one additional HA binding protein is a link module. Thelink module may be any link module described herein or known in the art.In some instances, the link module is a TSG6 link module, for example, ahuman TSG6 link module. In some instances, the link module of the HAbinding protein TSG6 may include amino acid residues 36-128 of humanTSG6 (UniProt Accession No. P98066).

Any of the preceding fusion proteins may specifically bind to VEGF andHA. In some instances, the fusion protein binds HA with a Kd of about 10pM or lower. For example, in some instances, the fusion protein binds HAwith a Kd of about 10 μM or lower, 8 μM or lower, 6 μM or lower, 4 μM orlower, 2 μM or lower, 1 μM or lower, 750 nM or lower, 500 nM or lower,250 nM or lower, 100 nM or lower, 50 nM or lower, 10 nm or lower, or 1nm or lower. In some instances, the fusion protein binds HA with a Kd ofabout 2 nM or lower. In some instances, the fusion protein binds HA witha Kd between about 1 nM and about 2 μM, for example, between about 1 nMand about 1.8 μM, between about 1 nM and about 1.6 μM, between about 1nM and about 1.4 μM, between about 1 nM and about 1.2 μM, between about1 nM and about 1.0 μM, between about 1 nM and about 900 nM, betweenabout 1 nM and about 800 nM, between about 1 nM and about 700 nM,between about 1 nM and about 600 nM, between about 1 nM and about 500nM, between about 1 nM and about 400 nM, between about 1 nM and about300 nM, between about 1 nM and about 200 nM, between about 1 nM andabout 100 nM, between about 1 nM and about 50 nM, between about 1 nM andabout 40 nM, between about 1 nM and about 30 nM, between about 1 nM andabout 20 nM, between about 1 nM and about 10 nM, between about 5 nM andabout 100 nM, between about 5 nM and about 50 nM, between about 5 nM andabout 25 nM, between about 5 nM and about 15 nM, or between about 5 nMand about 10 nM. In some instances, the fusion protein binds HA with aKd of about 10 nM.

Any of the preceding fusion proteins may have an ocular half-life thatis increased relative to a reference antibody that is not covalentlyattached to a HA binding domain. In some instances, the ocular half-lifeis increased at least about 2-fold (e.g., about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, about 12-fold, about 14-fold, about16-fold, about 18-fold, about 20-fold, or more) relative to thereference antibody. In some instances, the ocular half-life is increasedat least about 4-fold relative to the reference antibody. In someinstances, the ocular half-life is a vitreal half-life. In someinstances, the reference antibody is identical to the antibody of thefusion protein. In other cases, the reference antibody is non-identicalto the antibody of the fusion protein.

Any of the preceding fusion proteins may have an ocular clearance thatis decreased relative to a reference antibody that is not covalentlyattached to a HA binding domain. In some instances, the clearance isdecreased at least about 2-fold (e.g., about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold,about 18-fold, about 20-fold, or more) relative to the referenceantibody. In some instances, the clearance is decreased at least about4-fold relative to the reference antibody. In some instances, theclearance is clearance from the vitreous. In some instances, thereference antibody is identical to the antibody of the fusion protein.In other cases, the reference antibody is non-identical to the antibodyof the fusion protein.

In some instances, the time period between two intraocularadministrations (e.g., by intravitreal injection) of any of thepreceding fusion proteins is at least 1 month, e.g., at least 1 month,at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks,at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, atleast 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks,at least 48 weeks, at least 52 weeks or more. In some cases, the maximumperiod between two intraocular administrations is no longer then fouryears, e.g., no longer than three years, no longer than two years, or nolonger than one year. The fusion protein can be administered, forexample, every two to twelve months, e.g., every four to ten months. Insome instances, the fusion protein is administered every six months.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the fusion proteins described above.In certain embodiments, the composition comprises one or more additionalcompounds. In certain embodiments, the additional compound binds to asecond biological molecule selected from the group consisting of IL-1β;IL-6; IL-6R; PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrinsαvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin;complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; andproteins genetically linked to AMD risk, such as complement pathwaycomponents C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1;ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof. For example, in someinstances, the additional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the fusion proteins described aboveand an additional VEGF antagonist.

3. Polymeric Formulations

The invention provides formulations that include an anti-VEGF antibodyof the invention and a polymer. The polymeric formulation may be, forexample, a microsphere, an implant, a hydrogel, an organogel, anano-assembly, a micelle, an in situ forming depot, or another type ofpolymeric formulation known in the art. Any suitable polymer may be usedin the polymeric formulations of the invention. For example, the polymermay be a hydrophilic polymer or a hydrophobic polymer. It is to beunderstood that a hydrophilic polymer may be a water-soluble polymer.Any suitable hydrophilic polymer may be used, for example, a hydrophilicpolymer described in International Patent Application Publication No. WO2011/066417 or Pelegri-O'Day et al. J. Am. Chem. Soc. 136:14323-14332,2014. In other instances, hydrophobic polymers may be used, for example.The polymer may be biodegradable and/or biocompatible.

Exemplary, non-limiting hydrophilic polymers that can be used includehyaluronic acid (HA), polyethylene glycol (PEG; also known aspoly(ethylene glycol)) (e.g., straight-chain PEG, branched PEG,comb-like PEG, and dendritic PEG), poly[ethylene oxide)-co-(methyleneethylene oxide)], poly(poly(ethylene glycol) methyl ether methacrylate)(pPEGMA), agarose, alginate, carageenans, carboxymethylcellulose,cellulose, cellulose derivatives, chitosan, chondroitin sulfate,collagen, dermatan sulfate, dextran, dextran sulfate, fibrin,fibrinogen, fibronectin, fucoidan, gelatin, glycosaminoglycans (GAGs), aglycopolymer, heparin, heparin sulfate, a highly-branched polysaccharide(e.g., a galactose dendrimer), keratan sulfate, methyl cellulose,hydroxypropylmethylcellulose (HPMC),poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), pectins, pectinderivatives, pentosane polysulfate, starch, hydroxylethyl starch (HES),styrene, vitronectin, poly(acrylic acid), poly(methacrylic acid),poly(acrylamide), poly(acrylic acid), poly(amines), poly(amino acids),poly(carboxybetaine) (PCB), polyelectrolytes, poly(glutamic acid) (PGA),poly(glycerol) (PG) (e.g., linear, midfunctional, hyperbranched, orlinear hyperbranched PG), poly(maleic acid), poly(2-oxazoline) (POZ),poly(2-ethyl-2-oxazoline, polysialic acid (PSA), polystyrene,polystyrene derivatives (e.g., charged polystyrene derivatives),poly(styrenesulfonate-co-PEGMA), polyvinylpyrrolidone (PVP),poly(N-acryloylmorpholine) (pNAcM), and copolymers thereof. In otherinstances, any suitable hydrophobic polymer can be used, including, forexample, poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), andpolyglycolide (PGA).

The invention provides anti-VEGF antibodies formulated as polymersolvent depots (also known as in situ forming implants). For example, apolymer solvent depot may include a polymer (e.g., any of the polymersdescribed herein, e.g., a hydrophobic polymer such as PLGA), a solvent(e.g., an organic solvent), and an anti-VEGF antibody (e.g., anyanti-VEGF antibody described herein). The solvent may have a low watermiscibility. Exemplary organic solvents that may be used includetriacetin (glycerol acetate), N-methyl-2-pyrrolidone, poly(ethyleneglycol) dimethyl ether, and ethylbenzoate. In one working example, apolymer solvent depot can be prepared by dispersing a spray-dried powderof an anti-VEGF antibody (e.g., any anti-VEGF antibody described herein,e.g., G6.31 AARR) within a PLGA-triacetin solution (see, e.g., Chang etal. J. Pharm. Sci. 104(10):3404-17, 2015, which is incorporated hereinby reference in its entirety). The PLGA can have a molecular weight ofabout 10 kDa, about 41 kDa, or about 56 kDa, for example. PLGA polymersare commercially available, e.g., RG 752S, RG755S, and RG 756S (EvonikIndustries, Darmstadt, Germany). The PLGA concentration can be about7.5%, about 10%, about 12.5%, about 15%, about 20%, or more (% wt). Theantibody concentration can be about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 4%, about 5%, or more (% wt). In some instances,the antibody concentration is about 1.5% (% wt). Any of the polymersolvent depot formulations can be injectable, for example, through a 27gauge (27G) needle. Upon administration (e.g., by injection (e.g.,intravitreal injection)), such polymer solvent depot formulations cantransform into a gel or solid depot in the eye. The gel or solid depotmay form as a result of demixing of the aqueous and nonaqueous phases.Depending in part on the solvent transfer rate to aqueous phase, theinjected depot can transition to a solid or gel material because ofpolymer precipitation and physically entrap the anti-VEGF antibody (andany additional therapeutic agents or compounds). In other instances, insitu cross-linking or in situ-solidifying organogels, for example, canbe used to form a gel or solid depot. The polymer solvent depot mayallow long-term delivery, for example, over about 30 days or more, e.g.,about 30 days, about 40 days, about 50 days, about 60 days, about 70days, about 80 days, about 90 days, about 100 days, about 110 days,about 120 days, about 130 days, or more. In some cases, the polymersolvent depot may allow long-term delivery for about 80 days in the eye.

The invention also provides an anti-VEGF antibody (e.g., any anti-VEGFantibody described herein) formulated as a polymer micelle. A polymermicelle can be formed, for example, by self-assembly of a polymer (e.g.,an amphiphilic block (e.g., diblock or multiblock) copolymer) into ananoparticle having a hydrophobic core and a hydrophilic shell. Apolymer micelle may have any suitable diameter (e.g., average diameter),for example, a diameter of about 1 nm to about 1000 nm (e.g., about 1nm, about 10 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm,about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm,about 1000 nm), or larger. In some instances, a polymer micelle may havea diameter (e.g., average diameter) of about 5 nm to about 100 nm (e.g.,about 5, about 10, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, or about 100 nm). Any suitable polymer canbe used, including, for example, amphiphilic block copolymers such aspoly(propylene oxide) (PPO), poly(D,L-lactic acid) (PDLLA),poly(ε-caprolactone) (PCL), poly(L-aspartate), and poloxamers (e.g., apoly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethyleneoxide) copolymers (e.g., PLURONICS®)). Other amphiphilic blockcopolymers are known in the art. In some instances, the anti-VEGFantibody may be attached to the hydrophilic shell of the polymermicelle, for example, by a covalent attachment to the polymer.Accordingly, in some instances, an antibody conjugate described abovemay be used to prepare a polymer micelle that includes an anti-VEGFantibody.

The invention also provides an anti-VEGF antibody (e.g., any anti-VEGFantibody described herein, e.g., G6.31 AARR) formulated as a polymerimplant. A polymer implant is a rigid object in which a solid drugformulation (e.g., an antibody) is evenly distributed within ahydrophobic polymer (e.g., PLGA). A polymer implant is typicallymillimeter-sized (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm,0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm,1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.5 mm, 3mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, or larger). A polymerimplant may be administered to an eye, for example, by a surgicalprocedure (e.g., microsurgery) or by injection (e.g., by intravitrealinjection) using a suitable device. The polymeric implant can beformulated for administration to any suitable site in the eye, forexample, the vitreous, anterior or posterior chambers of the eye, orintraretinal, subretinal, intrachoroidal, suprachoroidal, episcleral,subconjunctival, intracorneal, or epicorneal sites of the eye. Inparticular instances, the polymeric implant is formulated foradministration to the vitreous.

A polymer implant that includes an anti-VEGF antibody (e.g., anyanti-VEGF antibody described herein, e.g., G6.31 AARR) can be preparedusing a hot-melt extrusion (HME) process. Heat is applied to melt(fluidize) the polymer, and then mechanical shear can be imparted toblend and microcompound a solid drug formulation within the fluidpolymer phase. The drug-polymer admixture can then be extruded through amillimeter-sized die (e.g., a circular die). When the extrudate isallowed to cool (e.g., to room temperature), it forms rods (e.g.,cyndrilical rods) that can be cut to any desired length. In someinstances, the polymer implant may be a PLGA rod that includes ananti-VEGF antibody and PLGA. Polymer implants that can be used in thecontext of the present invention are described, for example, inRajagopal et al. J. Pharmaceutical Sciences 102(8):2655-2666, 2013 andInternational Patent Application Publication No. WO 2006/093758, whichare incorporated herein by reference in their entirety.

In one working example, an anti-VEGF antibody (e.g., any anti-VEGFantibody described herein) can be prepared as a spray-dried formulation,which can include trehalose and histidine-HCl buffer for stability atelevated temperatures, which can then be added to a hydrophobic polymersuch as PLGA and undergo HME to form a polymeric implant (see, e.g.,Rajagopal et al. supra). For example, the spray-dried formulation mayinclude an antibody at any suitable concentration (e.g., 1 mg/mL, 2mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 20mg/mL, 25 mg/mL, 30 mg/mL), trehalose at any suitable concentration(e.g., 1 mg/mL, 2 mg/mL, 3 mg/mL, 3.3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL,7 mg/mL, 10 mg/mL, and/or a buffer (e.g., 10 mM histidine HCl). Thespray-dried formulation can have any suitable pH, for example, a pH ofabout between about 5 and about 8 (e.g., about 5, about 6, about 7, orabout 8). In some instances, the spray-dried formulation can have a pHof about 6 or about 6.2. In particular instances, the spray driedformulation includes an anti-VEGF antibody, 3.3 mg/mL trehalose, and 10mM histidine-HCl, pH 6.2. The polymeric implant may be prepared by HMEof the spray-dried formulation with a hydrophobic polymer such as PLGA.For example, solid PLGA pellets and the spray-dried formulation can bepremixed at room temperature and fed into a conical, counter-rotatingtwin-screw extruder. The combination can be microcompounded, followed byextrusion at 100° C. through a 0.5 mm circular die. In some instances,the spray-dried formulation is exposed to 100° C. for less than 30 min.

The polymer implant may contain, for example, from about 1% to about 90%anti-VEGF antibody by weight (e.g., about 1%, about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,or about 90%) by weight. In some instances, the polymer implant includesabout 10% anti-VEGF antibody by weight. The polymeric implant may haveany suitable dimensions, for example, in some instances, the implant hasa diameter of about 0.1 to about 1 mm (e.g., about 0.5 mm) and a lengthof about 1 to about 30 mm (e.g., about 14 mm). For PLGA implants (e.g.,PLGA rods), the percentage of polylactic acid in the PLGA copolymer canbe 0-100%, for example, about 15-85%, about 35-65%, or 50%. In someinstances, a PLGA implant (e.g., a PLGA rod) may further include one ormore additional polymers, for example, hydroxypropylmethylcellulose(HPMC).

In any of the preceding polymeric formulations (e.g., polymer solventdepots, polymer micelles, and polymer implants), the antibody may be anantibody fragment that binds VEGF. In some instances, the antibodyfragment is selected from the group consisting of Fab, Fab′, Fab-C,Fab′-SH, Fv, scFv, and (Fab)₂ fragments. For example, in some instances,the antibody fragment is an Fab, an Fab′, or a Fab-C.

Any of the preceding polymeric formulations (e.g., polymer solventdepots, polymer micelles, and polymer implants) may have an ocularhalf-life that is increased relative to a reference antibody that is notformulated as a polymeric formulation. In some instances, the ocularhalf-life is increased at least about 2-fold (e.g., about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about16-fold, about 18-fold, about 20-fold, or more) relative to thereference antibody. In some instances, the ocular half-life is increasedat least about 4-fold relative to the reference antibody. In someinstances, the ocular half-life is a vitreal half-life. In someinstances, the reference antibody is identical to the antibody of thepolymeric formulation. In other cases, the reference antibody isnon-identical to the antibody of the polymeric formulation.

Any of the preceding polymeric formulations (e.g., polymer solventdepots, polymer micelles, and polymer implants) may have an ocularclearance that is decreased relative to a reference antibody that is notformulated as a polymeric formulation. In some instances, the clearanceis decreased at least about 2-fold (e.g., about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, about 12-fold, about 14-fold, about16-fold, about 18-fold, about 20-fold, or more) relative to thereference antibody. In some instances, the clearance is decreased atleast about 4-fold relative to the reference antibody. In someinstances, the clearance is clearance from the vitreous. In someinstances, the reference antibody is identical to the antibody of thepolymeric formulation. In other cases, the reference antibody isnon-identical to the antibody of the polymeric formulation.

In some instances, the time period between two intraocularadministrations (e.g., by intravitreal injection) of any of thepreceding polymeric formulations is at least 1 month, e.g., at least 1month, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, atleast 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks,at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44weeks, at least 48 weeks, at least 52 weeks or more. In some cases, themaximum period between two intraocular administrations is no longer thenfour years, e.g., no longer than three years, no longer than two years,or no longer than one year. The polymeric formulation can beadministered, for example, every two to twelve months, e.g., every fourto ten months. In some instances, the polymeric formulation isadministered every six months.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the polymeric formulations (e.g.,polymer solvent depots, polymer micelles, and polymer implants)described above. In certain embodiments, the composition comprises oneor more additional compounds. In certain embodiments, the additionalcompound binds to a second biological molecule selected from the groupconsisting of IL-1β0, IL-6; IL-6R; PDGF; angiopoietin; angiopoietin 2;Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to AMD risk, such ascomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC; COL10A1; and TNFRSF10A. In certain embodiments, the additionalcompound is an antibody or antigen-binding fragment thereof. Forexample, in some instances, the additional compound is a bispecificantibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody, such asRG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecific antibodydisclosed in WO 2010/069532 or WO 2016/073157 or a variant thereof. Inanother example, in some instances, the additional compound is ananti-IL-6 antibody, for example, EBI-031 (Eleven Biotherapeutics; see,e.g., WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clazakizumab,sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921,or a variant thereof. In a still further example, in some instances, theadditional compound is an anti-IL-6R antibody, for example, tocilizumab(ACTEMRA®) (see, e.g., WO 1992/019579), sarilumab, vobarilizumab(ALX-0061), SA-237, or a variant thereof.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the polymeric formulations (e.g.,polymer solvent depots, polymer micelles, and polymer implants)described above and an additional VEGF antagonist.

4. Devices

Any of the compositions described herein (e.g., anti-VEGF antibodies,antibody conjugates, fusion proteins, and polymeric formulations) can beadministered to the eye using a port delivery device. A port deliverydevice is an implantable, refillable device that can release atherapeutic agent (e.g., an anti-VEGF antibody) over a period of months(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months). Exemplaryport delivery devices that may be used include those from ForSight Labs,LLC and/or ForSight VISION4, for example, as described in InternationalPatent Application Publication Nos. WO 2010/088548, WO2015/085234, WO2013/116061, WO 2012/019176, WO 2013/040247, and WO 2012/019047, whichare incorporated herein by reference in their entirety.

For example, the invention provides port delivery devices that includereservoirs containing any of the compositions described herein (e.g.,anti-VEGF antibodies, antibody conjugates, fusion proteins, andpolymeric formulations). The port delivery device may further include aproximal region, a tubular body coupled to the proximal region in fluidcommunication with the reservoir, and one or more outlets in fluidcommunication with the reservoir and configured to release thecomposition into the eye. The tubular body may have an outer diameterconfigured to be inserted through an incision or opening in the eye ofabout 0.5 mm or smaller. The device may be about 1 mm to about 15 mm inlength (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm inlength). The reservoir may have any suitable volume. In some instances,the reservoir has a volume of about 1 μl to about 100 μl (e.g., about 1μl, about 5 μl, about 10 μl, about 20 μl, about 50 μl, about 75 μl, orabout 100 μl). The device or its constituent parts may be made of anysuitable material, for example, polyimide.

In some instances, the port delivery device includes a reservoircontaining any of the compositions described herein (e.g., anti-VEGFantibodies, antibody conjugates, fusion proteins, and polymericformulations) and one or more additional compounds. In certainembodiments, the additional compound binds to a second biologicalmolecule selected from the group consisting of IL-1β; IL-6; IL-6R; PDGF;angiopoietin; angiopoietin 2; Tie2; SIP; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor; ST-2 receptor; and proteins genetically linkedto AMD risk, such as complement pathway components C2, factor B, factorH, CFHR3, C3b. C5, C5a, and C3a; HtrA1: ARMS2; TIMP3: HLA: IL-8: CX3CR1;TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments,the additional compound is an antibody or antigen-binding fragmentthereof. For example, in some instances, the additional compound is abispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody,such as RG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecificantibody disclosed in WO 2010/069532 or WO 2016/073157 or a variantthereof. In another example, in some instances, the additional compoundis an anti-IL-6 antibody, for example, EBI-031 (Eleven Biotherapeutics;see, e.g., WO 2016/073890), siltuximab (SYLVANT®), olokizumab,clazakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117,PF-04236921, or a variant thereof. In a still further example, in someinstances, the additional compound is an anti-IL-6R antibody, forexample, tocilizumab (ACTEMRA®) (see, e.g., WO 1992/019579), sarilumab,vobarilizumab (ALX-0061), SA-237, or a variant thereof.

In some instances, the port delivery device includes any of thecompositions described herein (e.g., anti-VEGF antibodies, antibodyconjugates, fusion proteins, and polymeric formulations) and anadditional VEGF antagonist.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Deep Scanning Mutagenesis of G6.31 Identifies PositionsImportant for Assembly of a Stable Assembled Fab

G6.31 is a high affinity anti-VEGF antibody (Fuh et al., J. Biol. Chem.281(10):6625-6631 (2006); Liang et al., J. Biol. Chem. 281(2):951-961(2006). G6.31 is considered representative of human antibodies as itsheavy and light chain variable domains are frequently used humangermlines IGHV3-23 and IGKV1-39 origin, respectively.

To systematically assess the effect of single mutations in the VH and VLdomains of G6.31, a saturated single site mutagenesis library for eachvariable domain (VL positions 2-107 and VH positions 2-113 according tothe Kabat numbering scheme) were generated. These libraries are referredto as the VH and VL NNK walk libraries, respectively. Using theselibraries in a deep mutational scanning experiment allowed thecalculation of an enrichment ratio (ER) for each mutation in the twolibraries during selection, which serve as an assessment of the impactof the mutation on the fitness of the molecule. The ER of a particularmutation aggregates a variety of effects, including the impact of themutation on functional expression, the impact of the mutation on thestability of the fold, and the impact of the mutation on binding to theselection protein (e.g., anti-gD (a peptide tag fused to the C-terminusof the light chain), protein A, protein L. or VEGF).

To assess the impact of each mutation on functional expression and foldstability of the Fab, the VH and VL libraries were panned separatelyagainst anti-gD, protein A, or protein L. These panning strategiesdetect whether a Fab molecule is displayed on phage and, to some extent,whether it is properly folded and assembled. The ERs of 2331 and 2226amino acid substitutions (including stops) and the respective wild-typeamino acid at a given position of the heavy chain library and lightchain library, respectively, were determined for each of the threepannings (FIGS. 1A-1F). A comparison of the three heavy chain panningstrategies or the three light chain pannings demonstrates that verysimilar results were obtained (FIGS. 2A-2B). For example, the ERs show agood linear relationship (r²=0.98) between anti-gD and protein L panning(FIG. 2A). A comparison between the protein A panning dataset and theprotein L panning dataset showed a lower linear correlation (r²=0.611),which can be attributed to the subset of mutations that have a directimpact on protein A binding and therefore a differential enrichment inthe two selections. A similar pattern was observed for the light chain(FIG. 2B).

In one approach to identify positions which impact the functionalexpression and stability of the Fab, the positions that were conservedand that do not tolerate mutations during the selection were determined.Using the data set obtained from anti-gD panning as a representativescan, the average enrichment ratio of all mutations at a given position(ER_(pos)) was calculated for each position as a measure of conservation(FIGS. 3A-3B). In both the VH and the VL domain, the central coreresidues, which consist of a cysteine pair (HC-C22, HC-C93; LC-C23,LC-88) between the n-strands B and F as well as an adjacent tryptophanresidue (HC-W36, LC-W35), were highly conserved (FIGS. 3A-3D). Thehydrophobic core of the immunoglobulin fold is described, for example,in Halaby et al. Protein Engineering 12(7): 563-571, 1999. Adjacent tothe central core are a few hydrophobic residues located in the lowerhalf of the molecule (when the Fab is orientated such that the HVRs faceupward) which are part of the extended hydrophobic core. They wereconserved but showed smaller ER_(pos) values than the central coreresidues. Among these residues is a tyrosine (HC-Y90, HC-86), which hasbeen shown previously to be important for the stability of the Ig-fold(see, e.g., Hamill et al., J. Mol. Biol. 295(3):641-649 (2000). Thethird group of conserved residues was located in the VH/VL interface(HC-L45, HC-Y91, HC-W103, LC-P44, LC-Y36, LC-Y96). VH/VL interfaceresidues were not only conserved in pannings with indirect selection(e.g., VH data from protein L panning), but also direct selection (e.g.,VH data from Protein A panning), indicating that mutations at thementioned interface positions resulted in an assembled Fab with reducedstability. The fourth group of conserved residues (HC-E46, HC-R38,HC-D86, LC-R61, LC-D82) includes residues in a hydrogen bond network atthe lower half of the Fab that are important for stability. Especiallythe aspartate residue in al-helix belongs to the positions, which showedone of the highest conservation in the dataset. Mutation at positionLC-R61 and LC-D82 have been shown previously to induce fibril formationand induce aggregation in light chains (see Helms et al., J. Mol. Biol.257(1):77-86 (1996)). Last, four positions in the HVR-H3 were identifiedas being highly conserved: HC-A93, HC-R94, HC-P100, HC-D101. Since thesepanning strategies were not selecting for antigen binding, these resultsindicated that the HVR3 loop conformation is involved in the stabilityof the Fab.

In summary, with the exception of a few positions of the variabledomains being highly conserved, many positions can tolerate mutationswithout affecting the overall stability and expression of the Fabmolecule in these selections. Further, even some positions that have asmall ER_(pos) can tolerate conserved amino acid substitutions. Forexample, the mutagenesis data suggested that hydrophobic residues of thelower core can be substituted with similar hydrophobic residue withoutaffecting the stability of the Fab (FIGS. 1A-1F). The tolerance of theVH/VL stability for single substitutions contrasts the results obtainedfrom a deep mutagenesis scan of the CH3 domain of a human IgG1, in whichthe majority of residues did not tolerate any mutations (see Traxylmayret al., J. Mol. Biol. 423(3):397-412 (2012)).

Materials and Methods

A. Full Variable Domain NNK Walk Library Design and Generation.

The full VH (residues 2-113) and VL (residues 2-107) of G6.31 weresubject to randomization. 10-12 subsequent residues were randomized persub-library. Within the sub-library, a TAA codon was introduced at eachrandomized position by Kunkel mutagenesis (Kunkel, Proc. Natl. Acad.Sci. USA 83(2):488-492 (1985)). 10 VL sub-library and 12 VH sub-librarystop templates were generated. For library design, each position wasrandomized by oligonucleotide-directed mutagenesis with an NNK codon,where N is any of the four natural nucleotides, and K is 50% T (thymine)and 50% G (guanine). The NNK codon can encode any of the 20 naturalamino acids. The sub-libraries for the light chain and heavy chain weremade separately and then the respectively libraries for each chain werecombined to form a VL library and a VH library. The combined librariesare also referred to as VH or VL NNK walk libraries. Libraries were madein a phage Fab fragment display vector. The VH NNK walk library had asize of 3×10⁹ members, while the VL NNK walk library had a size of 8×10⁸members.

B. Phage Panning Selection on Anti-gD. Protein L, or Protein a

Binding clones were selected by incubating the VH or VL NNK walk phagedisplay library with 5, 0.5, 0.1 nM biotinylated VEGF in successiverounds of selection, and then competed with 100 nM non-biotinylated VEGFat room temperature or 37° C. to reduce binding of the lower affinityclones to VEGF. Bound clones were captured on ELISA plates coated withneutravidin, washed, and eluted in 100 mM HCl for 20 minutes at roomtemperature. The eluted phage was neutralized with 1/10 volume of 1 MTris pH 8.0 and used to infect E. coli for amplification for the nextround of selection.

For selection with anti-gD, protein L, or protein A, binding clones wereselected by incubating the VH or VL NNK walk phage display library onELISA plates coated with anti-gD, protein L, or protein A, washed, andeluted in 100 mM HCl for 20 minutes at room temperature. The elutedphage was neutralized with 1/10 volume of 1 M Tris pH 8.0 and used toinfect E. coli for amplification for the next round of selection.

C. Illumina Sequencing of G6.31 Deep Mutational Scanning Libraries

For deep sequencing, phagemid DNA was isolated from E. coli XL1 cellscarrying phagemid vectors from either the unselected or the selected VHor VL NNK walk library. Purified DNA was used as template for a limitedcycle PCR-based amplification of VL and VH regions. PCR products werepurified by agarose gel extraction and clean-up (Qiagen Gel ExtractionKit). Eluted amplicon DNA was used as the basis for deep sequencinglibrary preparation with standard Illumina library preparation methods,using TRUSEQ™ DNA Sample Prep (Illumina). Adapter-ligated libraries weresubjected to a single cycle of PCR and sequenced on the Illumina MISEQ®,paired-end 300 bp to cover the entire length of the amplicon.

D. Deep Scanning Mutagenesis Data Analysis

Sequenced paired end reads were merged using FLASh (Magoc et al.,Bioinformatics 27(21):2957-2963 (2011)). Further sequencing dataanalysis was performed using the statistical programming language R(Team RC “R: A language and environment for statistical computing” RFoundation for Statistical Computing (2014)) and the ShortRead (Morganet al., Bioinformatics 25(19):2607-2608 (2009)) package. The first stepin quality control was filtering for sequences which carried therespective HC and LC barcode. In a second step, the flanking regions ofthe VH and VL domain were identified for each merged read and checkedfor the correct length in order to remove the vast majority of readswith insertion or deletion mutations (indels). To further correct forsequencing errors, non-NNK mutations were converted back to thewild-type base and reads which contained more than two NNK mutationswere filtered out. Position weight matrices were generated bycalculating the frequency of all mutations of every randomized position.Enrichment ratios for all mutations were calculated by dividing thefrequency of a given mutation at a given position in the sorted samplewith the frequency of the very same mutation in the unsorted sample, asdescribed previously (Fowler et al., Nature Methods 7(9):7412-746(2010)).

Example 2: Deep Scanning Mutagenesis Screen of G6.31 Identifies AminoAcid Residue Variants with Enhanced Stability and/or Improved BindingAffinity to VEGF

To obtain a comprehensive overview of the impact of single substitutionsof G6.31 on antigen binding, the VH and VL NNK walk libraries describedin Example 1 were panned against VEGF (FIGS. 4A-4B). The obtainedenrichment ratio (ER) values showed a bimodal distribution (FIGS.5A-5B). A subset of mutations had a strong negative effect on thebinding function, while the majority of mutations were neutral, and afew mutations had a strong positive effect on fitness. Mutations whichhad a negative impact on binding were for the most part located ineither the HC-HVRs, as those loops are considered to provide most of thebinding function of G6.31, or in the conserved residues of the frameworkas identified in the anti-gD panning (see Example 1). Strongly enrichedmutations were located mostly in the less-conserved section of theframework regions and the HC-HVRs. These results confirm the observationfrom the gD panning described above (see Example 1) that the variabledomains are robust and can tolerate many single mutations withoutsubstantially affecting the antigen-binding function of the Fab.

To confirm the results obtained from the deep mutagenesis scanning,selected mutations were expressed and purified, and their affinitytowards VEGF was measured using BIACORE® surface plasmon resonance (SPR)and their thermostability (as assessed by melting temperature, T_(m))was measured using differential scanning fluorimetry (DSF). The effectof selected strongly enriched mutations on antigen binding and foldingwas evaluated. Further, mutations with high enrichment were selectedbased on their occurrence at a given position in human antibodiesaccording to the Abysis database. In addition, some mutations which werestrongly depleted were also tested as negative controls. The Kd andT_(m) of the indicated G6.31 variants is shown in Table 2. The Kd valueslisted in Table 2 were measured using single cycle kinetic analysisusing a BIACORE® T200 device.

TABLE 2 Characterization of G6.31 Variants Identified By Deep ScanningMutagenesis Fold difference Melting k_(on) k_(off) Kd (relativeTemperature Clone (1/Ms) (1/s) (nM) to G6.31) (° C.) G6.31 WT 4.45E+056.86E−04 1.54 — 83.4 LC-Q3A 3.89E+05 3.59E−04 0.92 1.7 82.8 LC-M4Q5.93E+05 6.57E−04 1.11 1.4 72.8 LC-S7Q 7.31E+05 6.71E−04 0.92 1.7 83.2LC-S9Q 1.47E+06 6.55E−04 0.44 3.5 83.2 LC-S12M 8.94E+05 6.79E−04 0.762.0 82.9 LC-G16K 3.67E+05 3.56E−04 0.97 1.6 79.1 LC-T22D 4.87E+053.48E−04 0.72 2.1 81.8 LC-D28R 1.17E+06 6.55E−04 0.56 2.8 80.6 LC-Y36G2.25E+05 5.58E−04 2.47 0.6 66.8 LC-Q37A 6.03E+05 5.15E−04 0.85 1.8 81.6LC-S50M 4.67E+05 6.11E−04 1.31 1.2 84.5 LC-L73G 5.89E+05 6.44E−04 1.091.4 68.8 LC-F83A 8.55E+05 4.33E−04 0.50 3.1 88.8 LC-Q89N 9.91E+054.35E−04 0.44 3.5 79.6 LC-Q89T 1.17E+06 4.93E−04 0.42 3.7 79.1 LC-T97N4.32E+05 8.02E−04 1.86 0.8 81 LC-N94A 3.34E+05 1.31E−03 3.93 0.4 85.8LC-N94Q 2.78E+05 1.33E−03 4.78 0.3 84.6 HC-V2R 4.64E+05 3.41E−04 0.732.1 81.6 HC-S7L 7.36E+05 5.68E−04 0.77 2.0 83 HC-Q13M 1.26E+06 5.19E−040.41 3.8 83.1 HC-A40E 1.13E+06 4.66E−04 0.41 3.8 83 HC-I51H 2.48E+061.52E−03 0.62 2.5 83.1 HC-A53R 8.29E+05 3.50E−04 0.42 3.7 84.3 HC-T57E1.93E+06 4.02E−04 0.2 7.7 84.4 HC-Y58R 1.48E+06 2.55E−04 0.17 9.1 83.2HC-Y58Q 3.52E+05 3.58E−04 1.02 1.5 84.2 HC-Y59T 3.60E+05 9.74E−04 2.710.6 82.9 HC-A60M 3.86E+05 7.29E−04 1.89 0.8 82.8 HC-S70H 6.58E+056.60E−04 1 1.5 83 HC-T73N 6.78E+05 5.75E−04 0.85 1.8 83.1 HC-Q81S1.07E+06 4.87E−04 0.45 3.4 83 HC-Q81W 1.20E+06 5.42E−04 0.45 3.4 83.2HC- 1.39E+06 3.22E−04 0.23 6.7 83.1 N82aR

Using the data from 34 mutations (16 on the VH and 18 on the VL), nolinear relationship between the enrichment ratio and the obtained gainin affinity or stability was observed (FIGS. 5C-5D). Without wishing tobe bound by theory, this could reflect the fact that ER is influenced bya variety of factors including functional expression in E. coli,stability of the Fab on the phage particle, as well as binding to theselection protein. One example that illustrates this is the mutationLC-L73G, which is part of the hydrophobic core of the light chain.Replacing the hydrophobic leucine at this position with a glycine likelydecreases the packing in the core and leads to a less-stable lightchain. The T_(m) of the LC-L73G mutant was 14.4° C. lower that thewild-type G6.31 Fab. The LC-L73G mutation was depleted in the VEGFpanning experiment although it did not have a marked impact on antigenbinding affinity as measured by BIACORE® SPR (approximately 1.6-foldincrease (i.e., lower K_(d)) in affinity compared to wild-type G6.31).The depletion of G6.31LC_(L73G) was likely an effect of the reduceddisplay of the mutant on phage, and this effect dominated the selectionof this mutant over the actual antigen selection. Furthermore, theopposite is also true: a mutation which increases the display (forexample, by enhancing expression and/or stability) could be enrichedalthough the actual antigen binding function is impaired or not changedcompare to the wild-type (generating a false positive). Thereforevariants identified by enrichment may be improved for either bindingaffinity, stability, expression, and/or other properties.

Two mutations in HVR-H2 identified by the deep scanning mutagenesisapproach, HC-T57E and HC-Y58R, had the highest increase in affinity (8-to 9-fold) compared to wild-type G6.31 (FIG. 5E). A framework regionmutation, HC-N82aR, led to an approximate 6.6-fold affinity increasecompared to wild-type G6.31 (FIG. 5E). Several other framework regionmutations showed a 2- to 4-fold increase in affinity compared towild-type G6.31 (FIGS. 5E-5F). The highest increase in thermostabilitywas obtained in the light chain mutation LC-F83A (+5.4° C.) (FIG. 5F).In addition to the increase in melting temperature, LC-F83A increasedthe affinity of G6.31 to VEGF by 3-fold. Surprisingly, many of themutations which resulted in an increase in affinity were located distalto the antigen-binding site (FIGS. 5E-5F). Among these were HC-N82aR,which is located in the loop between helix al and strand β E, more than22 Å away from the antigen-binding site, and LC-F83A, which is locatedin the interface between the VL and the light chain constant domain(CL), more than 25 Å away from the HVRs.

In summary, the deep scanning mutagenesis of G6.31 using VH and VL NNKwalk libraries and next-generation sequencing identified amino acidresidue alterations that increased the binding affinity to VEGF and/orthe stability of the Fab.

Materials and Methods

A. Phage Panning Selection on VEGF

Binding clones were selected by incubating the VH or VL NNK walk phagedisplay library (described in Example 1) with 5, 0.5, 0.1 nMbiotinylated VEGF in successive rounds of selection, and then competedwith 100 nM non-biotinylated VEGF at room temperature or 37° C. toreduce binding of the lower affinity clones to VEGF. Bound clones werecaptured on ELISA plates coated with neutravidin, washed, and eluted in100 mM HCl for 20 minutes at room temperature. The eluted phage wasneutralized with 1/10 volume of 1 M Tris pH 8.0 and used to infect E.coli for amplification for the next round of selection.

B. Illumina Sequencing of G6.31 Deep Mutational Scanning Libraries andData Analysis

For deep sequencing, phagemid DNA was isolated from E. coli XL1 cellscarrying phagemid vectors from either the unselected or the selected VHand VL NNK walk libraries panned against VEGF. Purified DNA was used astemplate for a limited cycle PCR-based amplification of VL and VHregions. PCR products were purified by agarose gel extraction andclean-up (Qiagen Gel Extraction Kit). Eluted amplicon DNA was used asbasis for deep sequencing library preparation with standard Illuminalibrary prep methods, using TRUSEQ™ DNA Sample Prep (Illumina).Adapter-ligated libraries were subjected to a single cycle of PCR andsequenced on the Illumina MISEQ®, paired-end 300 bp to cover the entirelength of the amplicon. Data analysis was performed as in Example 1 tocalculate position weight matrices and enrichment ratios.

C. Antibody Expression

VH and VL sequences of selected variants were cloned into a mammalianFab vector for expression. Plasmids for both the heavy and light chainswere transfected (15 μg) into 30 ml 293T cells for 7 days. Thesupernatant was harvested to purify by protein G column.

D. Antibody Affinity Determinations by BIACORE® SPR

To determine the binding affinity of selected Fab variants, SPRmeasurement with a BIACORE® T200 instrument was used. Briefly, series Ssensor chip CM5 was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents according tothe supplier's instructions, and human VEGF (hVEGF) was coupled toachieve 50-80 response units (RU), then following by blocking un-reactedgroups with 1 M ethanolamine.

Three-fold serial dilutions of Fab in HBS-P buffer (0.01 M HEPES pH 7.4,0.15 M NaCl, 0.005% surfactant P20) from low (0.02 nM) to high (50 nM)were injected (flow rate: 30 μl/min). The binding responses on hVEGFwere corrected by subtracting of RU from a blank flow cell. Thesensorgram was recorded and subject to reference and buffer subtractionbefore evaluating by BIACORE® T200 Evaluation Software (version 2.0).Association rates (k_(on)) and dissociation rates (k_(on)) werecalculated using a simple one-to-one Langmuir binding model. Theequilibrium dissociation constant (Kd) was calculated as the ratiok_(off)/k_(on).

E. Melting Temperature (T_(m)) Determination by Differential ScanningFluorimetry

Differential scanning fluorimetry (DSF) monitors thermal unfolding ofproteins in the presence of a fluorescent dye and is typically performedby using a real-time PCR instrument (e.g., Bio-Rad CFX). SYPRO® Orangedye (Invitrogen, Cat. No. S6650) was diluted 1:20 in phosphate bufferedsaline (PBS). 1 μl of diluted dye was added into 24 μl Fab protein(approximately 100 μg/ml) per well. The temperature was increased from20° C. to 100° C. using a real-time PCR instrument (Bio-Rad CFX), andthe fluorescence intensity was plotted and the inflection point of thetransition curve (T_(m)) was calculated using equations such as theBoltzmann equation (see, e.g., Niesen et al., Nature Protocols2(9):2212-2221 (2007)).

Example 3: Conformational Changes of LC-F83 Correlate with the Fab ElbowConformation in GB Structures

To understand how mutations spatially remote from the antigen bindingsite could have such a strong impact on antigen binding and stability,the structural effect of the LC-F83A mutation was examined in moredetail. The parental antibody of G6.31, G6, has been crystallizedpreviously in the VEGF-bound and VEGF-free forms (Fuh et al., supra).G6.31 carries only four substitutions in the HVR-L3 compared to G6. Thecrystal structure of G6 was therefore used as a model for G6.31. Thecrystal structure of the VEGF-free form of G6 contains 12 Fab moleculesin the asymmetric unit. The Fab structures can be clustered into twodifferent groups based on the orientation of the constant domains to thevariable domains (V-C interface) (FIG. 6A), which is a result ofdifferent conformations in the Fab elbow region and can be quantified bymeasuring the Fab elbow angle (see, e.g., Stanfield et al., J. Mol.Biol. 357(5):1566-1574 (2006)).

The flexibility of the Fab elbow angle is influenced by a ball-socketjoint in the heavy chain (see, e.g., Lesk et al., Nature335(6186):188-190 (1988)), but also, and perhaps more importantly,dominated by the light chain class. While kappa light chain antibodiesare typically restricted in the elbow angle they can adapt and show abimodal distribution peaking at approximately 140° and approximately175°, rarely exceeding 180°, lambda light chain antibodies can adopt awider range of angles (see, e.g., Stanfield et al., supra). Six G6molecules in crystal structure have small elbow angles (143-155°) and atight packing of the DE-loop of CL domain against al of the VL, whilethe other six molecules have a large elbow angle (170-187°) with aloosely packed VL-CL interface (FIGS. 6B-6C). The interface areadistribution of different Fab molecules having a loosely packed or atight packed VL-CL interface is shown in FIG. 7C. The tight packedinterface area is, on average, approximately 600 Å, while the looselypacked interface area is approximately 450 Å. Further and more detailedanalysis revealed that six G6_(free) molecules have small elbow angles(143-155°) which is associated with a tightly packed VL-CL interface(comprising the DE-loop of CL domain packed against α1 of VL; 304±48Å²), whereas the other six have a large elbow angle (170-187°) with aless tightly packed VL-CL interface (234±29 Å² interface area). Thus,the G6 crystal structure reveals that G6 behaves like a typical humankappa light chain antibody with elbow angles below 180° (FIGS. 6A-6C).

The side chain conformation of LC-F83 correlated with the changes in theFab elbow angle. In the large elbow angle structures, LC-F83 is tightlybound in a hydrophobic pocket (referred to as the “in” conformation) inthe distal capping region of the VL domain (FIG. 6C). In the small elbowstructures, LC-F83 is flipped out (referred to as the “out”conformation) from the hydrophobic pocket and is partiallysolvent-exposed (FIG. 6B). Although located spatially close to the Fabelbow region, LC-F83 is not part of the elbow. However, LC-F83'sconformational changes are mirrored by changes in the side chainposition of the elbow residue LC-1106. In structures in which LC-F83 isthe ‘out’ conformation, the side chain of LC-F1061 moves “up” andoccupies the hydrophobic pocket. In contrast, in structures in whichLC-F83 is in an “in” conformation, the side chain of LC-1106 is moved“down” (note that “up” and “down” orientations are described withreference to the HVRs as being “up” and the constant domain being“down”). In contrast to LC-F83, the movement of LC-1106 is not limitedto the side chain and results also in conformational changes in theprotein backbone (LC-105 ϕ-angle, LC-106 ψ-angle), likely resulting inthe different elbow angles observed. In summary, the crystal structuresof G6 suggested that changes in the elbow angle in G6 requireconformational changes at position LC-1106 and LC-F83.

Example 4: The Coupling of Conformations of LC-83F with LC-1061 is aCommon Feature in Human Antibody Structures

G6/G6.31 originated from a phage library, which is built on commonframework regions for the heavy and the light chain (Lee et al., J. Mol.Biol. 340(5):1073-1093 (2004)). The closest germline genes for the lightchain are IGKV1-39 and IGKJ1. The most common used human IGKV subgroups,according to the IMGT database (Lefranc et al., In Silico Biology5(1):45-60 (2005)), IGKV1 and IGKV3 carry a phenylalanine at positionLC-83, while the other less frequently used germlines typically carry avaline (IGKV2 and IGKV4) or an alanine (IGKV5 and IGKV6) at position 83.The isoleucine at position LC-106 is conserved in all five human IGKJgermline genes.

To determine whether the coupling of the side chain conformation ofposition F83 with the Fab elbow conformation is a common feature inhuman antibodies, the conformation of LC-F83 was determined in 319 humanFab crystal structures from the Protein Data Bank (PDB). The structureswere grouped as structures with LC-F83 in an “in” conformation (dihedralangle chi1 between −50° and −100°) and structures in an “out”conformation (chi1 angle mostly between 500 and 180°) (FIG. 7A). TheLC-F83 conformation correlated in the vast majority of structures withchanges in the Fab elbow region at position LC-106 (FIG. 7B). Further,the two groups exhibited significant differences in the elbow angle(FIG. 7C) as well as in the area of the V-C interface, demonstratingthat structures with a large elbow angle have smaller, less tight packedV-C interface, while structures with a large elbow angle have a largeV-C interface (FIG. 7C).

Since the LC-F83A mutation resulted in higher thermostability and higheraffinity towards the antigen, the elbow angle of 20 human Fab structuresavailable in the PDB carrying LC-83A and LC-1061 was determined. Thesestructures mirrored the properties of the LC-83F “out” confirmationstructures: they exhibited a small elbow angle and large V-C interface(FIG. 7C).

In summary, these results demonstrated that the bimodal distribution inthe Fab elbow angle of human kappa light chain antibodies as observedpreviously (Stanfield et al., supra) is mainly a result of differentconformations of the phenylalanine side chain at position LC-83. Thedata further showed that the type of amino acid which is present atposition LC-83 has a large influence on the Fab elbow angle.

Materials and Methods

Human antibody structures were obtained from the structural antibodydatabase (SAbDab, Dunbar et al., Nucleic Acids Research 42:D1140-1146(2014)). Additional structure filtering and structure handling (aminoacid renumbering, dihedral angle determination, structural alignments)was performed using Bio3D (Grant et al., Bioinformatics 22(21):2695-2696(2006)). The interface area between constant and variable domains wascalculated using a local instance of CCP4-Pisa (Krissinel et al., J.Mol. Biol. 372(3):774-797 (2007)). ABangle (Dunbar et al., Protein Eng.Des. Sel. 26(10):611-620 (2013)) was used to characterize the VH-VLinteraction. Pymol and a publically-available script was used tocalculate the elbow angle.

Example 5: Molecular Dynamics Simulation Confirms that Position LC-83Acts Like Switch to Transition Between a Large and Small Fab ElbowConformation

Molecular dynamics was used to gain further insight into the effects ofthe LC-F83A mutation identified in the structural meta-analysisdescribed in Example 4. The effect of the LC-F83A mutation was simulatedin two different structural backgrounds. A G6 Fab crystal structure witha large elbow angle (G6 chains VU, “VU.F83”) and a crystal structurewith a small elbow angle (G6 chains BA, “BA.F83”) were used. Bothstructures in addition to the mutated structures (VU.F83A and BA.F83A)were simulated for 100 ns in water. During the time of the simulation,no changes in the conformation of F83 were observed: in the case ofVU.F83, LC-F83 stayed in an “in” conformation, while in case of BA.F83,the residue stayed in an “out” conformation (FIG. 8A). A comparison ofthe changes in the elbow angle over time during the simulation revealedthat the elbow angles of all four molecules stayed fairly stable duringthe simulation after an initial equilibration phase of about 25 ns (FIG.8B). No statistically significant difference in the elbow angledistribution of BA.F83 and BA.F83A was observed. For both molecules, theelbow angle fluctuated around 135°. However, there was a significantdifference between the elbow angle of VU.F83 and VU.F83A during thesimulation. During the first 25 ns of the simulation, the elbow angle ofVU.F83A collapsed and fluctuated around 140°, while the elbow angle ofVU.F83 stayed stable at a large angle of 161° (FIG. 9A).

The results presented above demonstrate the importance of the LC-F83“in” conformation to stabilize a large Fab elbow angle. The LC-F83Amutation resulted in an immediate transition to a small elbow angle anda larger VL-CL interface in the molecular dynamics simulation. Becauseno transition of the LC-F83 side chain from an “in” to an “out”conformation and visa versa could be observed, these results suggestthat LC-F83 imposes a significant energy barrier on changes in the elbowangle of the molecule.

Taken together, the increase in thermostability of G6.31 L-F3A comparedto G6.31 can be explained by an increase in the V-C interfaceinteraction area, which in turn further stabilizes the fold of the fourdomains of the Fab. Further, the tighter packing in the V-C interfacereduces the solvent exposure of hydrophobic residues like LC-1106.

Materials and Methods

Molecular dynamics simulations were performed using amber12 (Case etal., J. Comput. Chem. 26(16):1668-1688 (2005)). If not otherwise noted,100 ns were simulated at constant pressure and 300K. Before simulation,sections in the constant domains of the G6 structures which were notresolved because of missing electron density were complemented using thePDB entry 4hh9. The obtained simulated structures were analyzed usingVMD (Humphrey et al., Journal of Molecular Graphics 14(1):33-38, 27-38(1996)), Bio3d, CCP4-Pisa, Pymol and ABangle.

Example 6: Elbow Angle Dynamics of the G6 Fab Influence theAntigen-Binding Interface by Modulating the VH-VL Torsion Angle

While the change in Fab elbow angle explained the increase in meltingtemperature in the LC-F83A variant, the effect of this mutation onantigen binding was not directly explained by the change in Fab elbowdynamics. Without wishing to be bound by theory, the LC-F83A mutationcould influence antigen binding indirectly via the change in Fab elbowangle, or could influence antigen binding directly, as LC-F83A sitsclose to the VH-VL interface and could affect the orientation of the VHand VL domains towards each other. The VH-VL interface, although not indirect contact with the antigen, can have a strong influence in antigenbinding affinity (see, e.g., Masuda et al., FEBS J. 273(10):2184-2194(2006); Khalifa et al., Journal of Molecular Recognition 13(3):127-139(2000)). Further, in the antigen-free state, the VH-VL interface of aFab is typically flexible, with antigen binding resulting in an increasein rigidity (Dunbar et al., Prot. Eng. Des. Sel. 26(10):611-620 (2013)).In addition, the VH-VL orientation can vary substantially between theligand-free and the antigen-bound form (see, e.g., Stanfield et al.,Structure 1(2):83-93 (1993)).

Indeed the crystal structures of G6 showed flexibility and significantdifferences in VH/VL torsion angle (HL torsion angle) between differentG6 antigen-free structures (G6_(unbound)) and the VEGF-bound structures(G6_(bound)). The mean VH/VL torsion angle (HL torsion angle) of the sixG6 see molecules, which have a large elbow angle is approximately 60°,while it is approximately 58° for the six molecules which have a smallelbow. This is in turn substantially closer to the mean HL torsion angleof G6_(VEGF), which is −55° (FIG. 9B).

Molecular dynamics simulation was used to exclude the possibility thatthese changes in torsion angle were an artifact of crystallization. Themedian HL torsion angle for VU.F83 is approximately 62° while it wasapproximately 59° for BA.F83, conforming the results found inG6_(unbound) crystal structures. Moreover, the two mutant Fabs (BA.F83Aand VU.F83A) exhibited an even smaller torsion angle (approximately 57°and approximately 57°, respectively), which is even closer to thetorsion angle observed in G6_(bound) structures (FIG. 9C). Moreover, themutation at LC-F83A suggested an additional effect of the mutation onthe VH-VL interface, as the HL-angle of BA.F83A was significantlysmaller than that of BA.F83 (p<0.0001) These results indicated that theLC-F83A mutation influenced the HL torsion angle and in turn antigenbinding by two different mechanisms. First, the LC-F83A mutationmediates this indirectly by changing the Fab elbow angle. Structuraldata and molecular dynamics simulations showed that there is acorrelation between the elbow angle and the HL-angle (FIGS. 8A-8C and9A-9D). Second, LC-83 directly influences the VH-VL interface. There wasan additional significant increase in the HL torsion angle when LC-F83Awas introduced (FIGS. 9A-9D, compare molecule BA.F83 and BA.F83A). Theresult is an antigen binding site configuration in the antigen-free formof G6.31_(LC-F83A) which is closer to the one observed in the structureof G6_(VEGF). Without wishing to be bound by theory, the rearrangedantigen binding interface of G6.31_(LC-F83A) could result in lowerenergy cost for VEGF binding, and thus the observed increase in affinity(decreased Kd) of G6.31_(LC-F83A). Additionally, the presumably betterpacking of the VH-VL interface in G6.31_(LC-F83A) could provide analternative or additional fold stabilization mechanism (see, e.g.,Rothlisberger et al., J. Mol. Biol. 347(4):773-789 (2005)) to explainthe observed increase in T_(m).

Materials and Methods

Structural analysis and molecular dynamics simulations were performed asdescribed in Examples 3-5.

Example 7: Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)Confirms Conformational

Changes in the Constant Variable Interface as Well in the AntigenBinding Region in G6.31_(F83A) Compared to G6.31

HDX-MS was used to demonstrate that the changes in inter-domain dynamicsof the Fab molecule due to the LC-F83A mutation described in Examples3-6 can be observed in solution. HDX-MS measures the exchange rate ofbackbone amide hydrogen atoms with deuterium. Backbone amide hydrogenatoms typically exchange faster if they are solvent-exposed and notinvolved in the formation of hydrogen bonds compared to when they areburied and/or involved in the formation of hydrogen bonds. Severalregions show differences when the hydrogen-deuterium exchange pattern ofG6.31 is compared to G6.31_(LC-F83A) (FIG. 9D) The DE-loop of the CLdomain, which forms the VL-CL interface and is in proximity to theLC-F83A mutation, exchanged slower in G6.31_(LC-F83A) compared to G6.31.Changes in the exchange rate of HVR loops and neighboring regions werealso observed. The HVR loops H1 and L2 exchanged slower inG6.31_(LC-F83A) compared to G6.31, while the adjacent regions of theHVR-H3 loop which sit in the VH-VL interface exchanged faster. TheHVR-L2 and HVR-H3 loop are part of the VH-VL interface (see, e.g.,Vargas-Madrazo et al., Journal of Molecular Recognition 16(3):113-120(2003); Aburatani et al., J. Biochem. 132(5):775-782 (2002); Padlan,Molecular Immunology 31(3):169-217 (1994)). Therefore, the changes inthe exchange pattern observed in the HVRs and the VL-CL interface inHDX-MS between G6.31 and G6.31_(LC-F83A) independently confirmed theresults obtained from the structural analysis and molecular dynamicssimulation.

Materials and Methods

G6.31 WT and F83A mutant samples (45 μM) were diluted 15-fold into a 20mM histidine-acetate buffer at pD 7.0 and 50 mM NaCl with >90% D20content to begin the labeling reaction at 20° C. At sixlogarithmically-spaced time intervals spanning 30 seconds to 1000minutes, the labeling reaction was quenched by pH reduction (pH 2.5) andthe addition of 2M guanidinium chloride (GdmCl) and 0.25 Mtris(2-carboxyethyl)phosphine (TCEP) before injection onto a cold onlinesystem (see, e.g., Mayne et al. J. Am. Soc. Mass Spectrom.22(11):1898-1905, 2011).

Briefly, quenched samples were first passed through an immobilizedpepsin column (2.1×30 mm, Applied Biosystems) at 0° C. for proteolysisand then bound to a trap column for desalting (ACQUITY VANGUARD™ C8)before being separated by a reverse-phase chromatography (ACQUITY UPLC®BEH C18, 1.7 μm particle size, 1.0×50 mm) and introduced into the massspectrometer (Thermo ORBITRAP ELITE™, 120 k Hz resolution at m/z 400)for measurement of deuterium content. Chromatographic mobile phases wereprepared as described previously (Walters et al. J. Am. Soc. MassSpectrom. 23(12):2132-2139, 2012) with pH of 2.25 to maximize deuteriumrecovery which averaged 82% by measurement of fully deuterated controls.Mutant and wild-type experiments were interleaved and randomizedtimepoints were collected in triplicate; the entire process wasautomated by the LEAPv1 robotics platform (Leap Technologies, Carrboro,N.C.) which performed all sample handling steps. This process resultedin 152 unique peptides, consistently identified by the ExMS program (Kanet al. J. Am. Soc. Mass Spectrom. 22(11):1906-1915, 2011) for bothwild-type and mutant samples, providing sequence coverage of roughly95%. Analysis of deuterium content involved the use of previouslydescribed custom python scripts (Kan et al. supra; Walters et al. Proc.Natl. Acad. Sci. USA 110(47):18898-18903, 2013; and Hu et al. Proc.Natl. Acad. Sci. USA 110(19):7684-7689, 2013), and significantdifferences in deuterium uptake levels between wild-type and mutant wereidentified by Student's t-test as having a p-value <0.05, describedpreviously for HDX-MS experiments (Leurs et al. Anal. Chem.86(23):11734-11741, 2014).

Example 8: Somatic Hypermutation Target LC-83 and LC-106 in HumanAntibodies

Position LC-83 is located close to an AID hotspot motif (RGYW, where Ris a purine, Y is a pyrimidine, and W is A or T) in the IGKV1-39germline light chain gene. The increase in thermostability caused by theLC-F83A mutation was transferable to other antibodies which originatefrom the same germlines as G6.31. Therefore, the frequency of somaticmutations at position LC-83 in human antibodies was evaluated. Inaddition, LC-106 was also included in this analysis.

Two approaches were used to examine the distribution of somatichypermutation (SHM) in light chain sequences originating from IGKV1germlines. The first was to collate the somatic mutations of 4623 uniquehuman IGKV1 light chain sequences available in several publiclyavailable databases (FIG. 10A, upper panel). The second was byamplifying IGKV1 sequences from RNA originating from over 1000individual human lymphoid tissues followed by high-fidelity singlemolecule real-time sequencing (SMRT) and SHM collating. Despite thedifferent origin of the two datasets, a very similar distribution ofsomatic mutations was observed (FIGS. 10A-10C). LC-83 is one of the mostfrequently mutated positions in the VL segment; 7% and 19% of all IGKV1sequences carry a mutation at LC-83 in the two datasets, respectively.Interestingly, position LC-106 also frequently mutated in IGKV1sequences (9% and 20%, respectively), and the most common mutation isvaline. The closest germline gene segments for the G6 VL are IGKV1-39and IGJ1. Therefore, the antibodies of IGKV1.39 germline source wereexamined, which revealed that somatic mutations at LC-F83 primarilyresult in smaller side chains (including serine, valine, and leucine)(FIGS. 10A-10C). Alanine substitutions in antibodies of IGKV1.39 werenot observed, likely because an alanine mutation would require atwo-base substitution in the codon at LC-F83 that rarely occurs.

Next, G6.31 Fab variants carrying the most frequent somatic mutationsfor position LC-83 and LC-106 were generated, and their effect onantigen binding and thermostability was determined (FIG. 10D). Allsingle mutation variants increased the T_(m) by 4.8° C. to 6° C.,presumably by decreasing the Fab elbow angle and increasing the VL-CLinterface area. Consistently, the double mutation LC-F83A/LC-1106V didnot result in a significant increase in T_(m). Without wishing to bebound by theory, simultaneously reducing the size of both side chainswould leave a void in the hydrophobic cavity typically occupied byLC-F83 or LC-1106, and reduce the ability of the VL to stably packagainst the CL. The effect of the LC-83 mutation on VEGF affinity ismixed. LC-F83V leads to a 3-fold increase in affinity, similar toLC-F83A, whereas the mutations LC-F83S and LC-1106V showed nearly noincrease in affinity. The effect of the LC-83 mutation on antigenaffinity may be context dependent. LC-F83A mutations were introducedinto another antibody originated from the same germline as G6.31 and asimilar increase in thermostability and affinity was observed.Additionally, an LC-F83S mutation has been reported to increase theaffinity of an anti-estradial antibody.

In summary, LC-83 and LC-106 are frequently mutated in human antibodies.Given the influence of these two positions on the affinity andstability, optimization of the overall Fab domain dynamics is amolecular mechanism that may be highly utilized during antibody affinitymaturation in vivo.

Materials and Methods

Human light chain sequences were obtained from various publiclyavailable sources: Abysis (Available at the bioinf.org.uk/abysiswebsite), Kabat database (Martin, Proteins 25(1):130-133 (1996)), IMGTdatabase (Lefranc, Molecular Biotechnology 40(1):101-111 (2008)),GenBank (Benson et al., Nucleic Acids Research 43:D30-35 (2015)), andProtein Data Bank (Berman et al., Nucleic Acids Research 28(1):235-242(2000)). Duplicated sequences were removed and the closest germlinegenes were assigned for the respective V-segments using IGBlast (Ye etal., Nucleic Acids Research 41:W34-40 (2013)). Sequences for which amouse or rat germline was assigned were removed from the dataset.Sequences were Kabat numbered (see, e.g., Wu et al., J. Exp. Med.132(2):211-250 (1970)), and somatic mutations in the framework of theV-segment were identified. Mutations in the HVR regions (as defined bythe Kabat numbering schema) were not considered in this analysis.

The second source of human antibody sequences was from the commerciallyavailable RNA from human spleen, tonsil, bone marrow, and peripherialblood lymphocytes originated from 1088 individuals (Clonetech andAmsbio). The RNA was first transcribed into cDNA using the SMARTER® RACEcDNA Amplification Kit (Clonetech). IGKV-specific DNA was amplifiedusing 5′ RACE (Advantage 2 PCR Kit, Clonetech) with a custom primerannealing the 5′ region of the IGKC region(5′-CATCAGATGGCGGGAAGATGAAGACAGATGGTGC-3′ (SEQ ID NO: 56)), while IGKV1segments were enriched in a second PCR step using primers: forward:5′-GCCATCCAGATGACCCAGTCTCC-3′ (SEQ ID NO: 57) and reverse:5′-GGCTGCACCATCTGTCTTC-3′ (SEQ ID NO: 58). The PCR product was gelpurified and sequenced using Pacific Bioscience RSII (EA GenomicServices). A total of 994.8 Mbp were obtained. The consensus sequence ofeach read was created using Pacific Bioscience's SMRT Analysis 2.3software package. The germline annotation of the consensus sequences wasperformed using VDJFasta (Glanville et al. Proc. Natl. Acad. Sci. USA106(48):20216-20221). Sequences were Kabat numbered and somaticmutations in the framework of the V-segment were identified. In total,19034 sequences were annotated as IGKV1, of which 3796 contained atleast three framework regions and two HVRs and were included to generatethe SHM distributions.

Example 9: Identification of Surface Charge Variants of G6.31

To isolate mutations which could be used to lower the pI of G6.31,surface-exposed positions were identified within the variable domainsusing structural information from the G6 crystal structure. Positionsfor which a substitution to glutamate showed an enrichment ratio of 1 orgreater based on the VEGF panning of the NNK walk VH and VL libraries(Example 2) were selected to test their impact on VEGF binding affinityby single cycle kinetic analysis using a BIACORE® T200 system andthermostability (DSF) (Table 3A). Mutant variants containingsubstitutions to glutamate, as well as other charged residues (e.g.,arginine, lysine), were generated, expressed, and purified as describedabove (see Example 2, Materials and Methods). Note that, in someinstances, the overall Kd listed in Table 3A for some variants is weakeras compared to the results in Table 4 due to fitting differences betweensingle cycle kinetic analysis (Table 3A) and multiple cycle kineticanalysis (Table 4). Table 3B shows the yield, percent monomer (%monomer), and elution time for the indicated variants.

TABLE 3A Characterization of Surface-Exposed Charge Variants Fold changek_(on) k_(off) Kd (relative Variant (1/Ms) (1/s) (M) to G6.31) T_(m)G6.31 (WT) 3.01E+05 5.13E−04  1.7E−09 — 83.8 LC-S7E 3.56E+05 4.43E−041.24E−09 1.4 85.8 LC-S9E 1.24E+06 4.57E−04 3.68E−10 4.6 85.4 LC-S10E2.63E+05 4.29E−04 1.63E−09 1.0 86 LC-S12E 2.01E+05 4.58E−04 2.27E−09 0.784 LC-A13E 4.83E+05 3.94E−04 8.17E−10 2.1 79.2 LC-T20E 2.46E+05 4.57E−041.86E−09 0.9 84.6 LC-Q27E 2.30E+05 4.50E−04 1.96E−09 0.9 84.6 LC-P40E3.03E+04 5.41E−04 1.79E−08 0.1 83 LC-A51E 1.72E+05 4.62E−04 2.68E−09 0.676.8 LC-Y55E 4.53E+04 5.31E−04 1.17E−08 0.1 80.8 LC-G57E 4.29E+054.18E−04 9.74E−10 1.7 83 LC-P59E 2.84E+05 4.44E−04 1.56E−09 1.1 82.8LC-S77E 7.11E+05 3.99E−04 5.62E−10 3.0 84.6 LC-Y92E 4.97E+05 1.03E−032.07E−09 0.8 80 LC-G93E 2.48E+05 1.49E−03 5.98E−09 0.3 76.2 LC-A13R3.25E+05 4.58E−04 1.41E−09 1.2 81.6 LC-G16K 3.11E+05 4.46E−04 1.43E−091.2 79.6 LC-S50R 2.45E+05 4.64E−04 1.89E−09 0.9 84 LC-T31R 2.71E+054.89E−04 1.80E−09 0.9 84 LC-T72R 2.92E+05 4.46E−04 1.53E−09 1.1 84.4HC-V2E 3.00E+05 3.95E−04 1.32E−09 1.3 83.4 HC-S5E 7.88E+05 4.54E−045.76E−10 3.0 84 HC-S7E 7.61E+05 3.81E−04 5.01E−10 3.4 85.2 HC-G8E6.57E+05 4.55E−04 6.93E−10 2.5 83.4 HC-L11E 5.63E+05 5.07E−04 9.01E−101.9 82 HC-G16E 7.30E+05 4.54E−04 6.22E−10 2.7 83.2 HC-T28E 6.21E+053.52E−04 5.66E−10 3.0 85.2 HC-S30E 7.85E+04 5.58E−04  7.1E−09 0.2 84HC-A40E 1.97E+05 5.17E−04 2.63E−09 0.6 83.8 HC-P41E 1.52E+05 5.48E−04 3.6E−09 0.5 84.4 HC-K43E 3.80E+05 4.82E−04 1.27E−09 1.3 84.2 HC-Y58E8.20E+05 5.08E−04  6.2E−10 2.7 84 HC-A60E 5.24E+05 5.65E−04 1.08E−09 1.683.2 HC-K64E 5.52E+05 3.95E−04 7.15E−10 2.4 84 HC-G65E 3.02E+05 4.72E−041.56E−09 1.1 84.2 HC-T68E 2.15E+05 4.40E−04 2.05E−09 0.8 84.8 HC-A71E2.57E+05 3.79E−04 1.48E−09 1.1 84.8 HC-T73E 5.50E+05 4.34E−04 7.88E−102.2 84.6 HC-S74E 2.36E+05 4.99E−04 2.11E−09 0.8 84.4 HC-K75E 2.17E+054.44E−04 2.04E−09 0.8 84.2 HC-N76E 2.71E+05 4.67E−04 1.72E−09 1.0 81.4HC-T77E 2.48E+05 4.77E−04 1.93E−09 0.9 84.8 HC-Q81E 2.25E+05 4.95E−04 2.2E−09 0.8 85 HC-S82bE 4.37E+05 4.33E−04 9.91E−10 1.7 84.2 HC-Q105E1.09E+05 4.63E−04 4.24E−09 0.4 84.6 HC-T107E 4.16E+05 5.94E−04 1.43E−091.2 85.8 HC-L11R 8.90E+05 4.35E−04 4.88E−10 3.5 81.4 HC-T28K 9.75E+054.10E−04 4.20E−10 4.0 85.4 HC-P41R 5.71E+05 5.54E−04 9.70E−10 1.8 84.6HC-Y56K 7.24E+05 6.99E−04 9.65E−10 1.8 84.8 HC-T68K 3.97E+05 5.13E−041.29E−09 1.3 84.6 HC-T77K 2.03E+06 4.50E−04 2.22E−10 7.7 84

TABLE 3B Purification Parameters for Surface-Exposed Charge VariantsYield % Elution Time Variant (mg) Monomer (min) G6.31 (WT) 0.57 97.8612.74 LC-S7E 0.24 77.02 12.39 LC-S9E 0.32 77.19 12.43 LC-S10E 0.05 54.6412.44 LC-S12E 0.28 74.57 12.41 LC-A13E 0.31 67.58 12.4 LC-T20E 0.3273.31 12.36 LC-Q27E 0.26 78.98 12.19 LC-P40E 0.34 67.56 12.43 LC-A51E0.31 67.73 12.28 LC-Y55E 0.3 68.83 12.42 LC-G57E 0.3 67.9 12.28 LC-P59E0.35 71.85 12.41 LC-S77E 0.36 73.49 12.37 LC-Y92E 0.31 100 12.2 LC-G93E0.3 100 12.55 LC-A13R 0.35 70.15 12.41 LC-G16K 0.32 72.84 12.36 LC-S50R0.33 100 12.13 LC-T31R 0.34 74.87 12.49 LC-T72R 0.28 72.31 12.41 HC-V2E0.3 97.45 12.32 HC-S5E 0.36 97.14 12.44 HC-S7E 0.32 100 12.5 HC-G8E 0.33100 12.36 HC-L11E 0.33 100 12.48 HC-G16E 0.36 100 12.46 HC-T28E 0.2997.52 12.08 HC-S30E 0.36 94.81 11.9 HC-A40E 0.34 95.91 12.41 HC-P41E0.38 100 12.62 HC-K43E 0.36 100 12.54 HC-Y58E 0.37 100 11.97 HC-A60E0.27 97.58 12.39 HC-K64E 0.31 97.83 12.36 HC-G65E 0.32 98.01 12.41HC-T68E 0.35 100 12.41 HC-A71E 0.42 100 12.53 HC-T73E 0.37 100 12.14HC-S74E 0.36 100 12.36 HC-K75E 0.27 94.47 12.38 HC-N76E 0.37 95.68 12.02HC-T77E 0.41 97.5 12.51 HC-Q81E 0.33 97.72 12.49 HC-S82bE 0.4 93.5412.47 HC-Q105E 0.37 98.41 12.55 HC-T107E 0.32 98.04 12.23 HC-L11R 0.3799.81 12.53 HC-T28K 0.39 97.98 12.43 HC-P41R 0.34 100 12.6 HC-Y56K 0.39100 12.13 HC-T68K 0.42 100 12.39 HC-T77K 0.33 94.64 12.51

As shown in Table 3A, several surface-exposed charge variants wereidentified that had comparable or improved binding affinity to VEGF ascompared to G6.31.

Example 10: Generation and Characterization of Combination Variants withImproved Binding Affinity to VEGF and Improved Stability

A. Generation of Variants with Improved Binding Affinity to VEGF andImproved Stability

G6.31 contains an autocleavage site in HVR-L3 (N94-P95) which couldaffect its stability. Combinations of the single variants described inExample 2 (see, e.g., Table 2) were combined in order to identify cloneswith increased binding affinity and increased stability. Four mutationsin particular were focused on: LC-F83A (provides improvedthermostability and affinity), LC-N94A (removes the autocleavage site),HC-Y58R (improves affinity) and HC-N82aR (improves affinity). Severalother substitutions at position LC-N94 intended to remove theautocleavage site were also tested for their effect on binding affinityto VEGF by multiple cycle kinetic measurement as well as stability byDSF (Table 4). Removal of the autocleavage site consistently reducedfragmentation, as assessed by the percentage of low molecular weightentities determined using non-reduced capillary electrophoresis sodiumdodecyl sulfate (CE-SDS) (FIG. 12).

TABLE 4 Affinity and Thermostability of Auto-Cleavage Site VariantsT_(m) increase k_(on) k_(off) Kd T_(m) (relative Variant (1/Ms) (1/s)(nM) (° C.) to WT) G6.31 WT 7.54E+05 3.95E−04 0.525 84 — LC-N94A1.59E+06 1.69E−03 1.06 85.6 1.6° C. LC-N94Q 3.18E+05 3.62E−04 1.14 84.60.6° C. LC-N94R 1.94E+05 4.07E−04 2.09 85.2 1.2° C.

Combination of the LC-F83A, LC-N94A, HC-Y58R, and HC-N82aR variants intoa single clone (“Y58R.N94A.N82aR.F83A,” also referred to as “G6.31AARR”) resulted in a markedly increased binding affinity for VEGF (Kd=80pM), a 6.6-fold improvement compared to G6.31. Y58R.N94A.N82aR.F83A alsohad a markedly improved thermostability compared to G6.31, with a 4.8°C. increase in T_(m). (Table 5). The amino acid sequence of the VHdomain of Y58R.N94A.N82aR.F83A is shown in SEQ ID NO: 11, and the aminoacid sequence of the VL domain is shown in SEQ ID NO: 12.

TABLE 5 Combination variant Y58R.N94A.N82aR.F83A has improved bindingaffinity to VEGF and improved stability compared to G6.31 Fold T_(m)k_(on) koff Kd improved T_(m) increase Variant (1/Ms) (1/s) (nM)(relative to WT) (° C.) (relative to WT) G6.31 WT 7.54E+05 3.95E−040.525 — 84.0 — LC-F83A 1.65E+06 2.18E−04 0.132 4.0 89.0 5.0° C. LC-N94A1.59E+06 1.69E−03 1.06 0.5 85.6 1.6° C. HC-Y58R 2.42E+06 1.84E−04 0.0766.9 83.2 0.8° C. HC-N82aR 1.62E+06 1.24E−04 0.077 6.8 84.0 0.0° C.Y58R.N94A.N82aR.F83A 4.00E+06 3.20E−04 0.080 6.6 88.8 4.8° C.Y58R.N94A.F83A 2.22E+06 3.01E−04 0.135 3.9 89.2 5.2° C.

B. Generation of Surface-Exposed Charged Variants with Altered pI

To generate variants with lower pI, several glutamate substitutions wereintroduced into the sequence of G6.31. Based on the singlesurface-exposed charged variant mutation data (Tables 3A-3B) variantswhich did not have a large negative impact on VEGF binding were selectedfor further engineering. The expression level (as assessed by yield) andreduced aggregation (as assessed by high % monomer) were also consideredin selecting mutations. The iso-electrostatic surface of G6.31 wascalculated using APBS (Baker et al. Proc Natl. Acad. Sci. USA98(18):10037-10041 (2001)). Selected glutamate mutations were mappedonto the electrostatic surface and combinations of glutamate mutationswere chosen which (i) avoided mutations which were located spatiallyclose together and (ii) which favored mutations which were locatedwithin a strong positively-charged patch. Two heavy chain combinationvariants (with and without R19E) and five light chain combinationvariants were generated for further characterization (Table 6). Alllight chain combination variants contained the LC-N94A mutation toremove the auto-cleavage site and the LC-F83A mutation to improve thethermostability of the antibody. Several of the variants showed improvedaffinity to VEGF (see Table 6, Table 7, and Table 9). For example, HCLC2and HCLC5 showed improved affinity and a markedly reduced pI compared toG6.31 (pI 5.3 and 5.6, respectively, compared to pI 8.9 for G6.31) (seeTable 7 and Table 9).

TABLE 6 Combination Surface-Exposed Charge Variants SEQ Variant Type IDNO: Mutations HCcombo VH 33 V2E, S7E, R19E, T28E, A40E, G16E, K43E,T57E, K64E, K75E, S82BE, T107E R19HCcombo VH 51 V2E, S7E, T28E, A40E,G16E, K43E, T57E, K64E, K75E, S82BE, T107E LCcombo1 VL 34 S9E, R18E,F83A, N94A LCcombo2 VL 35 R18E, K42E, S76E, F83A, N94A LCcombo3 VL 36S9E, R18E, K42E, F83A, N94A LCcombo4 VL 37 R18E, F83A, N94A LCcombo5 VL12 F83A, N94A

TABLE 7 Combination Surface-Exposed Charge Variants Binding Kinetics toVEGF Antibody k_(on) k_(off) Kd Name (1/Ms) (1/s) (M) VH VL HCcombo2.05E+06 2.74E−04 1.34E−10 HCcombo G6.31 WT HCLC2 2.12E+06 6.23E−042.93E−10 HCcombo LCcombo2 HCLC4 2.03E+06 6.08E−04 2.99E−10 HCcomboLCcombo4 HCLC5 1.81E+06 5.73E−04 3.16E−10 HCcombo LCcombo5 HCLC31.84E+06 6.08E−04 3.30E−10 HCcombo LCcombo3 HCLC1 1.37E+06 6.07E−044.42E−10 HCcombo LCcombo1 G6.31 WT 9.99E+05 5.99E−04 5.99E−10 G6.31 WTG6.31 WT LCcombo2 5.99E+05 6.67E−04 1.11E−09 G6.31 WT LCcombo2 LCcombo35.74E+05 7.68E−04 1.34E−09 G6.31 WT LCcombo3 LCcombo5 5.37E+05 7.66E−041.43E−09 G6.31 WT LCcombo5 LCcombo4 4.63E+05 8.08E−04 1.75E−09 G6.31 WTLCcombo4 LCcombo1 4.47E+05 1.01E−03 2.27E−09 G6.31 WT LCcombo1 R19HCLC28.89E+05 4.67E−05 9.49E−11 R19HCcombo LCcombo2 R19HCLC4 2.84E+054.79E−05 1.69E−10 R19HCcombo LCcombo4 R19HCLC5 7.07E+05 4.41E−056.24E−11 R19HCcombo LCcombo5

In addition, several other combination variants were generated thatincluded selected surface-exposed glutamate substitutions and singlevariants that improved affinity and/or stability (Table 8).

TABLE 8 Binding Kinetics of Additional Combination Variants k_(on)k_(off) Kd Variants (1/Ms) (1/s) (M) HC-A40E, HC-T57E 2.96E+06 8.04E−042.72E−10 LC-F83A, LC-N94A 6.48E+05 1.11E−03 1.71E−9  LC-F83A, LC-N94A,LC-T22D 1.33E+06 7.44E−04 5.58E−10 HC-A40E, HC-T57E, LC-F83A, 1.27E+067.41E−04 5.84E−10 LC-N94A HC-A40E, HC-T57E, LC-F83A, 1.20E+06 7.18E−045.97E−10 LC-N94A, LC-T22D

Table 9 shows a summary of binding affinity, stability, and pI data forselected combination variants. Antibody pI was calculated using anin-house program and by running an isoelectric focusing (IEF) gel withstandard pI control. Table 10 shows the corresponding VH and VL aminoacid sequences for these antibodies. Table 11 shows the VL HVR aminoacid sequences for these antibodies. Table 12 shows the VH HVR aminoacid sequences for these antibodies.

TABLE 9 Summary of Selected Combination Variants Antibody k_(on) k_(off)Kd Fab Tm Name (1/Ms) (1/s) (nM) (° C.) pI G6.31 7.54E+05 3.95E−04 0.52583.8 8.9 LC-N94A 1.56E+06 1.69E−03 1.060 85.8 8.9 LC-N94A.LC-F83A6.48E+05 1.11E−03 1.710 88 8.9 LC-N94A.LC-F83A. 1.27E+06 7.41E−04 0.58088 8.5 HC-A40E.HC-T57E (G6.31 AAEE) N94A.F83A.N82aR.Y58R 2.22E+063.01E−04 0.135 88.2 9.3 (G6.31 AARR) HCcombo 2.05E+06 2.74E−04 0.13471.2 5.6 HCLC2 2.12E+06 6.23E−04 0.293 72.6 5.3 HCLC4 2.03E+06 6.08E−040.299 73.4 5.4 HCLC5 1.81E+06 5.73E−04 0.316 73.8 5.6 HCLC3 1.84E+066.08E−04 0.330 73.8 5.3 HCLC1 1.37E+06 6.07E−04 0.442 73.6 5.4 R19HCLC28.89E+05 4.67E−05 0.095 74.8 5.7 R19HCLC4 2.84E+05 4.79E−05 0.169 76.25.8 R19HCLC5 7.07E+05 4.41E−05 0.062 76.2 6

TABLE 10 VH and VL Amino Acid Sequences for Antibodies from Table 9Antibody Variant VH Variant VL Name (SEQ ID NO) (SEQ ID NO) G6.31 WTG6.31 WT G6.31 WT (SEQ ID NO: 42) (SEQ ID NO: 38) LC-N94A G6.31 WT N94A(SEQ ID NO: 42) (SEQ ID NO: 41) LC-N94A.LC-F83A G6.31 WT N94A.F83A (SEQID NO: 42) (SEQ ID NO: 12) LC-N94A.LC-F83A. A40E.T57E N94A.F83AHC-A40E.HC-T57E (SEQ ID NO: 40) (SEQ ID NO: 12) (G6.31 AAEE)N94A.F83A.N82aR.Y58R N82aR.Y58R N94A.F83A (G6.31 AARR) (SEQ ID NO: 11)(SEQ ID NO: 12) HCcombo HCcombo G6.31 WT (SEQ ID NO: 33) (SEQ ID NO: 38)HCLC2 HCcombo LCcombo2 (SEQ ID NO: 33) (SEQ ID NO: 35) HCLC4 HCcomboLCcombo4 (SEQ ID NO: 33) (SEQ ID NO: 37) HCLC5 HCcombo N94A.F83A (SEQ IDNO: 33) (SEQ ID NO: 12) HCLC3 HCcombo LCcombo3 (SEQ ID NO: 33) (SEQ IDNO: 36) HCLC1 HCcombo LCcombo1 (SEQ ID NO: 33) (SEQ ID NO: 34)R19HCcombo R19HCcombo G6.31 WT (SEQ ID NO: 51) (SEQ ID NO: 38) R19HCLC2R19HCcombo LCcombo2 (SEQ ID NO: 51) (SEQ ID NO: 35) R19HCLC4 R19HCcomboLCcombo4 (SEQ ID NO: 51) (SEQ ID NO: 37) R19HCLC5 R19HCcombo N94A.F83A(SEQ ID NO: 51) (SEQ ID NO: 12)

TABLE 11 VL HVR Sequences for Antibodies from Table 9 Antibody NameHVR-L1 HVR-L2 HVR-L3 G6.31 WT RASQDVSTAVA SASFLYS QQGYGNPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 23) LC-N94A RASQDVSTAVASASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)LC-N94A.LC-F83A RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) LC-N94A.LC-F83A. RASQDVSTAVA SASFLYSQQGYGAPFT HC-A40E.HC-T57E (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)(G6.31 AAEE) N94A.F83A.N82aR.Y58R RASQDVSTAVA SASFLYS QQGYGAPFT(G6.31 AARR) (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCcomboRASQDVSTAVA SASFLYS QQGYGNPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 23) HCLC2 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) HCLC4 RASQDVSTAVA SASFLYS QQGYGAPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCLC5 RASQDVSTAVA SASFLYSQQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCLC3RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10) HCLC1 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) R19HCcombo RASQDVSTAVA SASFLYS QQGYGNPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 23) R19HCLC2 RASQDVSTAVASASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) R19HCLC4RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10) R19HCLC5 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10)

TABLE 12 VH HVR Sequences for Antibodies from Table 9 Antibody NameHVR-H1 HVR-H2 HVR-H3 G6.31 WT DYWIH GITPAGGYTYYADSVKG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 53) (SEQ ID NO: 3) LC-N94A DYWIHGITPAGGYTYYADSVKG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 53)(SEQ ID NO: 3) LC-N94A.LC-F83A DYWIH GITPAGGYTYYADSVKG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 53) (SEQ ID NO: 3) LC-N94A.LC-F83A. DYWIHGITPAGGYEYYADSVKG FVFFLPYAMDY HC-A40E.HC-T57E (SEQ ID NO: 1)(SEQ ID NO: 21) (SEQ ID NO: 3) (G6.31 AAEE) N94A.F83A.N82aR.Y58R DYWIHGITPAGGYTRYADSVKG FVFFLPYAMDY (G6.31 AARR) (SEQ ID NO: 1) (SEQ ID NO: 7)(SEQ ID NO: 3) HCcombo DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) HCLC2 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) HCLC4 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1)(SEQ ID NO: 22) (SEQ ID NO: 3) HCLC5 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) HCLC3 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) HCLC1 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1)(SEQ ID NO: 22) (SEQ ID NO: 3) R19HCcombo DYWIH GITPAGGYEYYADSVEGFVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) R19HCLC2 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) R19HCLC4 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) R19HCLC5 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3)

The upper hinge region of the Fab heavy chain of any of the antibodieslisted above, for example, G6.31 AARR, can be mutated to removereactivity to anti-IgG1 hinge autoantibodies that has been reported inthe literature. See, e.g., Brezski et al., J. Immunol. 181:3183-3192,2008 and Brezski et al., mAbs 2:3, 212-220, 2010. Thus, the C-terminalamino acid of G6.31 AARR heavy chain can be either a T (wild-type (WT)version) or L (variant version that lacks reactivity to anti-human IgGFab). The full-length heavy chain amino acid sequence of wild-type G6.31AARR is SEQ ID NO: 48. The full-length heavy chain amino acid sequenceof the variant version that lacks reactivity to anti-human IgG Fab isSEQ ID NO: 49. The full-length light chain amino acid sequence for bothG6.31 AARR and the variant version that lacks reactivity to anti-humanIgG Fab is SEQ ID NO: 50.

In summary, combinations of variants identified by deep scanningmutagenesis resulted in antibodies with improved properties. In someinstances, the antibodies had improved binding affinity to VEGF as wellas improved stability (as judged by markedly increased T_(m)) ascompared to G6.31. Some of the variants (e.g., the antibodies HCcombo,HCLC1, HCLC2, HCLC3, HCLC4, and HCLC5) had both improved bindingaffinity to VEGF and markedly reduced pI. For the variants with reducedpI, reverting the R19E mutation in the heavy chain back to the originalarginine at position 19 (e.g., as in R19HCcombo, R19HCLC2, R19HCLC4, andR19HCLC5), resulted in variants with further affinity improvement andincreased T_(m) (by about 2.2-2.8° C.).

Example 11: Ocular and Systemic Pharmacokinetics of G6.31 VariantsFollowing Intravitreal Administration in Rabbits

To assess the pharmacokinetic (PK) properties of G6.31 variants in vivo,the following experiment was performed using New Zealand White (NZW)rabbits. Ocular half-life was determined for two different G6.31variants, G6.31 AAEE (LC-N94A.LC-F83A.HC-A40E.HC-T57E) and G6.31 AARR(Y58R.N94A.N82aR.F83A), as well as the parent G6.31 WT. In each case,Fabs of each of the antibodies were formulated in PBS at a concentrationof 10 mg/mL. 50 μL (0.5 mg) was injected intravitreally intoanesthetized rabbits. The rabbits (in groups of 10) were dosed once/eye.Group 1 was dosed with G6.31 AAEE, Group 2 was dosed with G6.31 WT, andGroup 3 was dosed with G6.31 AARR. Tissues (the vitreous humor, theaqueous humor, and serum) were collected pre-dose, at 2 hours, 6 hours,1 day, 2 day, 4 days, 8 days, 15 days and 21 days post-dose. Sampleswere assayed for the level of anti-VEGF using the Total Fab ELISAdescribed below.

The results of the pharmacokinetic analysis of each variant and WT ineach of the vitreous humor and aqueous humor are presented in FIG. 11A.The results show that the PK of G6.31 AAEE and of G6.31 AARR areessentially the same as the PK for WT G6.31 in both the vitreous humorand the aqueous humor. The vitreous and aqueous half-lives werecalculated and those results are presented in Table 13 below. Thehalf-lives of the variant G6.31 Fabs were similar to WT and also wereconsistent with ocular half-lives of Fabs reported in the literature.

TABLE 13 Half-life of G6.31 variants and G6.31 WT Vitreous Half-lifeAqueous half-life Antibody (days) (days) G6.31 AAEE 3.13 3.02 G6.31 WT3.23 3.03 G6.31 AARR 3.1 2.81

Systemic exposure to Fabs following intravitreal injection was assessedby measuring the levels of anti-VEGF in the serum. Serum samples wereassayed for anti-VEGF. The results are shown in FIG. 11B. The clearance(ml/kg/day) was calculated and those results are shown in Table 14below. Serum was also assayed for anti-therapeutic antibodies (ATA). ATAwas present in all animals from day 14 on.

TABLE 14 Clearance of G6.31 variants and G6.31 WT Clearance Antibody(ml/kg/day) G6.31 AAEE 733 G6.31 WT 1238 G6.31 AARR 2133

As shown by the results shown in Table 14, if all three Fabs have thesame bioavailability, then G6.31 AARR had the fastest clearance, whichindicates a lower systemic exposure compared to G6.31 WT and to G6.31AAEE. Such a lower systemic exposure may result in a better safetyprofile for G6.31 AARR compared to G6.31 WT and GG6.31 AAEE.

Materials and Methods

A. Total Fab ELISA

ELISA plates were coated with AffiniPure F(ab′)₂ fragment goatanti-human IgG overnight at 4° C. Plates were then washed 3 times priorto incubation with blocking buffer (PBS pH 7.4, 0.5% BSA, and 15 ppmPROCLIN™). After 3 washes, aqueous and vitreous samples collected fromNZW rabbits dosed with WT G6.31, G6.31 AARR, or G6.31 AAEE wereincubated in the plates for 2 h at room temperature under gentleagitation. The G6.31 molecules that bound to the coating antibodies werethen detected with an F(ab′)₂ peroxidase conjugated goat anti-human IgGfor 1 h at room temperature. After 3 washes, the TMBE-1000 substratesolution was added to the plates for 30 min and the reaction was stoppedusing 1M H₃PO₄. The signal was recorded at 450/620 nm.

Concentrations of G6.31 molecules were determined using a standardcurve.

Example 12: VEGF-Induced HUVEC Migration Assays

G6.31 variants were tested for the ability to inhibit VEGF-induced HUVECmigration. HUVEC migration assays of the variants indicated in Table 15were performed using Falcon 24-multiwell insert systems (BD Biosciencescat. 351184). The inserts were pre-coated with 8 mg/ml mouse laminin(LifeTechnologies 23017-015) overnight. HUVECs were starved overnight,harvested, and resuspended in assay medium (EBM-2, 0.1% BSA). Cells(5×10⁴) were added to the upper chamber, and 20 ng/mL of VEGF was addedto the lower chamber to stimulate migration in the presence or absenceof various dose levels of blocking antibodies for 16 h. After fixing andscraping from the upper face membrane, cells on the lower face werefixed with methanol and stained with SYTOX® green (LifeTechnologiesS7020). Images were acquired using an inverted fluorescent microscope,and cell number was analyzed using ImageJ software.

FIG. 13 shows a plot of inhibition of VEGF-induced HUVEC migration byG6.31 LC-N94A compared to the parent G6.31 at varying Fabconcentrations. As can be seen from FIG. 13, the single LC-N94A mutationwas significantly less potent, by about five-fold, in the VEGF-inducedHUVEC migration assay compared to the WT G6.31 parent. As shown in Table15, the double mutant, LC-N94A.LC-F83A was also about five-fold lesspotent in this assay compared to the WT G6.31 parent. The quadruplemutant, LC-N94A.LC-F83A.HC-A40E.HC-T57E (G6.31 AAEE) restored andslightly improved potency (Table 15). Surprisingly, the quadruplemutant, N94A.F83A.N82aR.Y58R (G6.31 AARR) had significantly improvedpotency, by about two-fold, compared to the WT G6.31 parent (Table 15).

TABLE 15 Cell potency of G6.31 variants determined by IC50 in HUVECMigration Assay Fab IC50 relative to G6.31 G6.31 1 LC-N94A 5.2LC-N94A.LC-F83A (AA) 5.1 LC-N94A.LC-F83A. 0.9 HC-A40E.HC-T57E (G6.31AAEE) N94A.F83A.N82aR.Y58R 0.5 (G6.31 AARR) ranibizumab 0.9

Example 13: Conjugation of Hyaluronic Acid (HA) to G6.31 AARR and OtherAntibodies

Current approaches for treatment of ocular disorders associated withpathological angiogenesis (e.g., AMD (e.g., wet AMD), DME, DR, or RVO)typically involve intravitreal injection of VEGF antagonists (e.g., theanti-VEGF Fab ranibizumab). Because the site of action of anti-VEGF Fabsis in the back of the eye at the retina, and also because Fabs can haverelatively short residence time in the eye, maximum patient benefit fromanti-VEGF Fabs is typically obtained by relatively frequent dosings(e.g., Q4W) by intravitreal injection. Long-acting delivery of anti-VEGFantibodies or antibody fragments (e.g., Fabs) for ocular disorders maybe desired, at least in part, to decrease dosing frequency, whichresults in improved patient convenience and compliance. In this Example,the effects of conjugating linear, uncrosslinked hyaluronic acid (HA) tothe G6.31 variant G6.31 AARR were evaluated. The molecular properties ofthe conjugates, pharmacokinetic parameters (e.g., vitreal half-life andclearance), thermodynamic stability, and VEGF inhibitory potency wereanalyzed.

A model rabbit Fab (rabFab; Shatz et al. Mol. Pharmaceutics 2016; PubMedidentifier (PMID) 27244474) was used as part of this analysis.Development of delivery technologies for protein therapeutics typicallyinvolves testing in relevant animal models to demonstrate in vivoutility. Rabbit models are commonly employed during early studies ofocular pharmacokinetics. Unfortunately, most human and humanizedantibodies are immunogenic in rabbits, precluding estimation of keypharmacokinetic parameters using long-acting delivery technologies. Toaddress this problem a surrogate compound was developed, which is aspecies-matched rabbit Fab (“rabFab”) that is useful for evaluatingdelivery technologies in rabbit models. The rabFab described herein wasderived from a rabbit monoclonal antibody that binds to human phosphoc-Met. Methods of making rabbit antibodies, including rabbit monoclonalantibodies, are well known in the art. See, for example, U.S. Pat. Nos.5,675,063 and/or 7,429,487. An exemplary rabbit monoclonal antibody thatbinds to human phospho c-Met is commercially available from Abcam(Cambridge, Mass., USA), product number ab68141.

In the present experiments, G6.31 AARR or rabFab Fab-C molecules wereconjugated to linear, uncrosslinked HA of various weight-average molarmasses (Mw), including 40 kDa (HA40K-), 100 kDa (HA100K-), 200 kDa(HA200K-), and 600 kDa (HA600K-). Fab-C molecules are Fab molecules thatare expressed such that the sequence is truncated at the first hingecysteine, resulting in a Fab with a free cysteine directly fromexpression. Fab′ molecules, which are Fab molecules with a free cysteinegenerated by digestion of a full-length monoclonal antibody, can also beused. For the HA conjugates described in this Example, the Fab-Cmolecules were covalently attached to carboxylic acid groups on theglucuronic acid saccharide units of HA. In the first step of synthesis,a certain percentage of these acid groups on HA were converted tomaleimides (typically 2-10% of acid groups were converted). The Fab-Cmolecules were then conjugated to the maleimide groups through the freecysteine on the Fab-C molecule. However, slight modifications to theconjugation chemistry can be used to conjugate Fab molecules or otherantibody formats to HA.

A combination of size exclusion chromatography (SEC), refractive index(RI) multi-angle light scattering (MALS), and quasi-elastic lightscattering (QELS) (also referred to as SEC-RI-MALS-QELS) was used toassess HA Mw, conjugate Mw, percentage Fab loading (defined as thepercentage of HA carboxylic acids groups which are occupied by acovalently-attached Fab molecule), hydrodynamic radius (Rh), free Fab(defined as the percentage of Fab molecules which are free in solutionand not covalently attached to a HA molecule), and protein mass fraction(protein mass/(protein mass+HA mass) of selected HA-Fab conjugates(Table 16). FIG. 14 shows exemplary results of SEC-RI-MALS-QELS toassess the weight-average molar mass (Mw) of HA40K-rabFab,HA200K-rabFab, and HA600K-rabFab (note that the QELS data is not shownin FIG. 14).

TABLE 16 Properties of select HA-Fab conjugates as assessed bySEC-RI-MALS-QELS Conjugate Protein HA Mw Mw % Fab Rh Free mass Sample(kDa) (kDa) loading (nm) Fab fraction HA40K- 45.3 636.2 7.4 ~10* 0.45%0.929 rabFab HA100K- 110.0 679.3 5.9  17.3 3.71% 0.838 rabFab** HA200K-204.3 1805.3 8.6 ~30* 0.55% 0.887 rabFab HA600K- 619.8 2569.2 3.2 ~50*1.87% 0.759 rabFab HA200K- 204.3 2478.0 11.0  27.7 0.63% 0.918 G6.31AARR *Approximated Rh values **Sample investigated in rabbit PK study(see, e.g., FIG. 16 and below)

For treatment of ocular disorders such as AMD, a long ocular half-lifebut short systemic half-life may be desired, for example, to minimizesystemic anti-VEGF exposure. HA clearance from systemic circulation ismediated by hyaluronidase-bearing hepatic cells. Previous reportsindicate that hyaluronidase activity on native HA involves recognitionthrough the glucuronic acid carboxylic acid groups. Since thesecarboxylic acid groups were modified to attach to the Fab-C molecules,the ability of the conjugated HA to be enzymatically digested byhyaluronidase was assessed. HA-conjugated rabFab retained enzymaticsusceptibility to digestion by hyaluronidase-2 (HYAL2), as assessed bySEC-MALS analysis of HYAL2-incubated HA and HA100K-rabFab (FIG. 15).

The effect of HA conjugation of Fab fragments on ocular pharmacokineticparameters (e.g., half-life and clearance) in rabbit vitreous followingintravitreal administration was determined. Conjugation of rabFab toHA100K (HA100K-I¹²⁵-rabFab) resulted in a significant decrease inclearance and a significant increase in vitreal half-life compared tounconjugated rabFab or ranibizumab (FIG. 16 and Table 17).HA100K-conjugated rabFab had an approximate four-fold increase invitreal half-life (t_(1/2)) compared to unconjugated rabFab fromhistorical data (11.9 days versus 3.2 days, respectively) (FIG. 16 andTable 17). Table 17 also shows the Rh for rabFab and HA100K-I¹²⁵-rabFab.These data indicate that HA conjugation increased the hydrodynamicradius of Fabs and resulted in slowed vitreal clearance and increasedvitreal half-life.

TABLE 17 Clearance of rabFab and HA100K-¹²⁵I-rabFab from rabbit vitreousafter intravitreal administration Rh t_(1/2) CL Test Article (nm) (days)(mL/day) HA100K-I¹²⁵-rabFab 17.3 11.9 0.076 rabFab (historical) 2.5 3.20.32

The linear correlation between hydrodynamic size (e.g., in terms ofhydrodynamic radius) and vitreal residence time (e.g., in terms ofhalf-life), along with historical data for rabFab, rabFab conjugated to20KDa polyethylene glycol (PEG) (rabFab-20 kDa PEG), and rabFabconjugated to 40 kDa PEG (rabFab-40 kDa PEG) (Shatz et al. supra),coupled with the data described above for HA100K-rabFab, allowedprediction of the vitreal half-lives of G6.31 conjugated to HA100K orHA200K and G6.31 conjugated to HA300K (FIG. 17). HA100K-G6.31 has apredicted vitreal half-life of approximately 11 days, while HA200K-G6.31has an even greater predicted vitreal half-life of approximately 17 days(FIG. 17). HA300K-G6.31 has a predicted vitreal half-life ofapproximately 19 days (FIG. 17). These results provide further evidencethat conjugation of G6.31 AARR or other G6.31 variants described hereinto linear, uncrosslinked HA results in increased ocular residence time(e.g., half-life) and decreased clearance following intravitrealinjection.

The effect of HA conjugation on Fab thermodynamic stability was alsoevaluated (Table 18). Covalent attachment of rabFab to HA of threedifferent molar masses did not alter the thermodynamic stability of theFab, as demonstrated by the similar Tm onset and peak Tm for conjugatescompared to the parent Fab (Table 18).

TABLE 18 Conjugation of Fabs to HA does not significantly alter Fabthermodynamic stability Sample Tm Onset, ° C. Tm, ° C. rabFab 66.8 85.6HA39K-rabFab 69.5 84.1 HA100K-rabFab 69.8 84.0 HA300K-rabFab 69.7 83.8

Finally, the effect of HA conjugation on G6.31 AARR's VEGF inhibitionpotency was assessed. Surprisingly, conjugation of G6.31 AARR to linear,uncrosslinked HA resulted in significantly improved potency over theparent Fab when compared in a pKDR assay for VEGF inhibition (Table 19).The qAC50 values in Table 19 indicate the concentration required toelicit a 50% response in the activity assay format. The qAC50 values areequivalent to IC50 values. The HA-conjugated G6.31 AARR had anapproximate 7-fold increase in potency compared to the unconjugatedparent Fab (Table 19). Therefore, conjugation of G6.31 AARR and otherG6.31 variants described herein can be useful both to improve vitrealpharmacokinetic parameters (including half-life and clearance) as wellas VEGF inhibitory potency.

TABLE 19 Conjugation of G6.31 AARR to HA results in improved VEGFinhibition potency Test article qAC50, nM G6.31.AARR 0.9325 HA100K-G6.31AARR 0.1346

Based on the foregoing experimental data, HA-conjugated G6.31 AARRmolecules have significantly improved characteristics for treatment ofocular disorders, including AMD (e.g., wet AMD), DME, DR, and RVO. Theseimproved characteristics include increased potency and the opportunityfor less-frequent dosing due to enhanced vitreal residence time.

Materials and Methods

A. Synthesis of Maleimide-Functionalized HA (HA-Mal)

Linear, uncrosslinked HA polymers of various molar masses were obtainedfrom Lifecore Biomedical. HA was modified with maleimide groups using anaqueous reaction with the coupling reagent4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM) and the linker N-(2-aminoethyl)maleimide trifluoroacetate salt(AEM). HA was dissolved in 100 mM 2-(N-morpholino)ethanesulfonic acid pH5.5 at 2.5 mg/mL and to this solution was added 1.5 molar excess DMTMMand 3.75 molar excess AEM (molar excesses are calculated based on molesof carboxylic acid in HA) under stirring. The solution was heated to 70°C. for 2 hours.

Excess AEM and DMTMM were removed from the reaction via a desaltingprocedure. A HiPrep™ 26/10 desalting column was mounted on an ÄKTA™purification system and equilibrated with 10 mM sodium acetate pH 4.0150 mM NaCl. The reaction was injected neat onto the column, and theHA-mal peak was collected according to absorbance at 302 nm andconcentrated to greater than 5 mg/mL using centrifugal ultrafiltrationdevices. The maleimide concentration in the HA-mal stock solution wasmeasured by absorbance at 302 nm using a UV-visible spectrophotometer,and the molar ratio of maleimide groups per HA chain was assessed viasize exclusion chromatography with multi-angle light scattering(SEC-MALS).

B. Conjugation of Fab-C to HA-Mal

A solution of Fab-C was pH adjusted to 6.5 using 1 M phosphate pH 6.5 toa final phosphate concentration of 50 mM. Ethylenediaminetetraaceticacid (EDTA) was spiked into the solution to a final concentration of 2.5mM. The Fab-C solution was stirred and to it was added HA-mal dilutedinto reaction buffer (10 mM phosphate 150 mM NaCl 2.5 mM EDTA (pH 6.5)).The stoichiometry was set at 1.2 moles of Fab-C per mole of maleimide inthe final reaction and the volume was set to give a final proteinconcentration of 1 mg/mL. The conjugation reaction was carried out atroom temperature under stirring. At 3 hours, mercaptoethanol was addedat 2 moles per mole of maleimide to cap unreacted maleimide groups.After 30 minutes following addition of mercaptoethanol, the reaction wasdiluted to less than 50 mM NaCl with 10 mM phosphate (pH 6.5) forpurification.

Purification was carried out using anion exchange chromatography toseparate free Fab-C and Fab dimer from the conjugate. A HiTrap™ Q FF 5mL column was mounted on an ÄKTA™ purification system and equilibratedwith 10 mM HisHCl (pH 5.5). The diluted reaction was flowed over thecolumn, capturing the HA-Fab conjugate and simultaneously eluting freeFab-C and dimer. After washing with 10 mM HisHCl (pH 5.5) 100 mM NaCl,the conjugate was eluted by a step gradient to 10 mM HisHCl (pH 5.5) 500mM NaCl. The eluted conjugate was pooled and diluted with 10 mM HisHCl(pH 5.5) to a final composition of 10 mM HisHCl (pH 5.5) 150 mM NaCl.The formulated conjugate was concentrated by centrifugalultrafiltration.

C. Analysis of HA-Fab Conjugates

Residual free Fab content, total conjugate molar mass, and protein massfraction were assessed by the combination of size exclusionchromatography (SEC) with refractive index (RI) multi-angle lightscattering (MALS), and quasi-elastic light scattering (QELS) (alsoreferred to as SEC-RI-MALS-QELS) on an Agilent 1200 high performanceliquid chromatography (HPLC) System with a Wyatt Optilab T-rEX RIdetector and Wyatt HELEOS-II MALS detector in-line. A Tosoh G6000 PWxlcolumn was used for analysis with PBS (pH 7.4) as the running buffer. ABSA control was used to normalize MALS detectors and correct for bandbroadening between detectors. Free Fab content was measured byintegrating the UV A₂₈₀ peaks corresponding with Fab and HA-Fabconjugate. Conjugate molar mass was taken as the weight-averagemolecular weight (Mw) (also referred to as weight-average molar mass) ofthe conjugate peak. Protein mass fraction was calculated using a proteinconjugate analysis using the differential RI (dRI) and UV A₂₈₀ signals.

D. Dynamic Scanning Calorimetry (DSC) Studies of HA-rabFab Conjugates

Solutions of rabFab, rabFab conjugated to HA having a weight-averagemolar mass (Mw) of 39 kDa (HA39K-rabFab), rabFab conjugated to HA havinga Mw of 100 kDa (HA100K-rabFab), and rabFab conjugated to HA having a Mwof 300 kDa (HA300K-rabFab) were prepared at 0.5 mg/mL (concentrationgiven on protein basis) in 10 mM HisHCl (pH 5.5) 150 mM NaCl. DSCstudies were carried out on a VP-DSC micocalorimeter (MicroCal, Inc.)from 15 to 105° C. with a 1° C./min ramp rate. A reference scan ofbuffer alone was subtracted from each sample scan. Melting temperature(Tm) and Tm onset were evaluated using the accompanying ORIGIN softwaresuite.

E. Phosphorylated KDR (pKDR) Receptor Activation Assays

CHO cells engineered to overexpress KDR (KDR-CHO cells; seeBinétruy-Toumaire et al. EMBO J. 19(7):1525-1533, 2000 and Benzinger etal. BBA Biomembranes 1466:71-78, 2000, which are incorporated herein byreference in their entirety) were plated at 1×10⁴ cells/well in 80 μl ofcell plating medium (50:50 High Glucose DMEM/Ham's F-12, 0.2% BSA, 0.25%diafiltered fetal bovine serum (FBS), 25 mM HEPES, and 2 mM L-glutamax)in a flat-bottom 96-well tissue culture plate. The cells were incubatedovernight at 37° C. In parallel, an NUNC® MAXISORB® 384-well ELISA plate(Thermo Catalog No. 439454) was coated with 25 μl of anti-gD antibody(Genentech) diluted in PBS, and incubated at 4° C. overnight.

The next day, 2 hours before cell stimulation, the tissue culture plateswere flicked and tamped, and the culture medium was changed into 40 μlof serum-free cell stimulation medium (50:50 High Glucose DMEM/Ham'sF-12, 0.5% BSA, and 25 mM HEPES) and incubated for 2 h at 37° C.Three-fold serial dilutions of G6.31 AARR or HA100K-G6.31 AARR were madein cell stimulation medium. Dilutions of human VEGF₁₆₅ (100 ng/ml) werealso prepared in cell stimulation medium. Using a deep well block plate,the samples from the serial dilutions of G6.31 AARR or HA100K-G6.31 AARRwere mixed with the diluted VEGF samples and incubated for 1 h at 37° C.40 μL of the resulting mixtures were added to each well of cells for atotal culture volume of 80 μL. Wells having VEGF only or no VEGF addedwere also prepared as controls. The cells were incubated for 15 min at37° C. The culture medium was flicked and tamped, and 25 μL of ice-coldcell lysis buffer (150 mM NaCl, 50 mM HEPES, 0.5% TRITON™ X-100, 1×HALT® protease and phosphatase inhibitor cocktail (Thermo Catalog No.78444, added immediately before use from 100× stock), 5 mM EDTA) wasadded. The cells were lysed on ice for 15 min before proceeding with theELISA.

The wells of the anti-gD coated ELISA plate were washed 3 times withwashing buffer (PBS with 0.05% TWEENS 20, pH 7.4). The wells wereblocked with 80 μl blocking buffer (PBS with 0.5% BSA (R&D Systems,Catalog No. #DY995)) for 1 h at room temperature. The wells were thenwashed three times with washing buffer. Next, 25 μl of cell lysate perwell was added to each well and incubated for 2 h at room temperature,followed by washing the wells four times with washing buffer. 25 μl of1:2000 diluted anti-phosphotyrosine (Clone 4G10) biotin-conjugatedantibody (0.5 μg/ml) (Millipore Catalog No. 16-103) was added in assaybuffer (PBS with 0.5% BSA; same as blocking buffer) to each well and theresulting mixtures were incubated for 2 h at room temperature, followedby three washes with washing buffer. 25 μl of 1:10,000 dilutedhorseradish peroxidase (HRP)-Strepavidin (GE Health Care UK; Catalog No.RPN44OIV) was added in assay buffer to each well and incubated for 30min at room temperature, followed by three additional washes withwashing buffer. 25 μl of TMB was then added to each well, and color wasallowed to develop for 20 min prior to addition of 25 of H₂SO₄ to stopthe reaction. Finally, the optical density of the wells were read on aplate reader at 450/620 nm.

F. Hyaluronidase Digestion of HA100K-rabFab

To confirm that Fab conjugation up to 10% (percent expressed on basis ofavailable acid groups on HA) did not alter the hyaluronidasesusceptibility of HA-Fab conjugates, HA with a Mw of 100 kDa (HA100K)and rabFab conjugated to HA100K (HA100K-rabFab) were incubated at 200μg/mL HA (HA100K-rabFab concentration was adjusted to achieve 200 μg/mLconcentration with respect to the HA backbone) with 4 μg/mLhyaluronidase-2 in 10 mM sodium acetate, pH 4.5. Samples were incubatedat 37° C. and were immediately injected onto SEC-RI-MALS at 30-minintervals for Mw analysis, as described above.

G. PK Study to Assess Rate of HA-Fab Clearance from Rabbit Vitreous

An ocular pharmacokinetic profile of HA100K-rabFab in rabbit vitreouswas compared to historical data for rabFab as described below.

HA100K-rabFab was labeled with ¹²⁵I on the day prior to dosing. Toprepare the dosing formulations, an appropriate amount of HA100K-rabFabwas exchanged with Tris iodination buffer (25 mM Tris HCl, 0.4 M NaCl,pH 7.5) using Millipore AMICON® 30K centrifugal filter tubes. A Piercepre-coated iodination tube was wetted with Tris iodination buffer anddecanted. An appropriate volume of Tris iodination buffer was added tothe bottom of the tube followed by an appropriate amount of Na¹²⁵I(Perkin Elmer). The iodine was allowed to activate for 15 min withswirling every 30 sec. An appropriate volume of test sample, Trisiodination buffer, and activated iodine were added to a NUNC®microcentrifuge tube and mixed by gentle shaking for approximately 1 to5 min. The iodination reaction was terminated by adding scavengingbuffer (tyrosine, 2 mg/mL Tris buffer), and the formulation was mixedand incubated for 5 min with flick mixing at 1 and 4 min. For proteinpurification, an AMICON® 30K centrifugal filter tube was prepared bycentrifugation at 6,000 RPM for 15 min with PBS. The NUNC® tube contentswere added to an AMICON® 30K centrifugal filter tube and washed up tofour times in PBS to achieve approximately 400 μL final radiolabeledtest article volume. The radioactivity, protein concentration, andradiochemical purity of each test article were determined. Using apipette, the contents of the AMICON® tube were transferred to a NUNC®tube and the formulation was brought to a final concentration ofapproximately 10 mg/mL.

HA100K-rabFab-¹²⁵I was injected intravitreally (500 μg/eye) into eyes ofNew Zealand White rabbits (n=24 eyes). For the intravitreal injections,rabbits were sedated/anesthetized to effect with isoflurane and placedin lateral recumbency. Tropicamide ophthalmic solution (two drops) and2.5% phenylephrine HCl (one drop) were applied to both eyes. Topicalproparacaine was applied to each eye immediately prior to preparationand dosing. The conjunctival fomices were flushed with a 1:50 dilutionof betadine solution and the eyelid margins were swabbed with undiluted5% betadine solution. The superotemporal bulbar conjunctiva was swabbedwith undiluted betadine solution. A Jameson caliper was used to mark aspot 1.5 to 2.0 mm posterior to the limbus on the superotemporal bulbarconjunctiva. Conjunctival forceps were used to fix the globe positionwith the left hand while the needle affixed to the injection syringe wasinserted with the right hand, at the marked spot, through the sclera andadvanced 5 mm into the vitreous humor. The injection needle waspositioned to face the posterior axis of the globe and the contents weredelivered into the mid-vitreous by slowly depressing the syringeplunger. The needle affixed to the injection syringe was disinserted andthe episcleral tissues approximated to the site of insertion graspedwith the conjunctival forceps for 30 sec to lessen reflux of theinjected material. The dose site was swabbed for residual radioactivity.Preparation and dose administration was repeated in the same manner forthe second eye. The appropriate test article was maintained on wet icefor the duration of the procedure and was administered once at a dosevolume of 50 μL per eye. Indirect ophthalmoscopic examinations wereperformed following injection to ensure no lens or retinal contactoccurred. A dose wipe of each injection site was collected and wasretained at room temperature prior to analysis by liquid scintillationcounting (LSC) to determine residual radioactivity. The radioactivityrecovered was subtracted from the administered amount to give the actualradioactive dose administered.

Blood samples were collected from the jugular vein of the animals intotubes containing no anticoagulant at room temperature pre-dose, 6 hourspost-dose, and 1, 2, 4, 7, 11, 14, 21, and 28 days post-dose. An aliquot(approximately 0.1 mL) of whole blood was collected and processed forradioanalysis. The remaining blood was centrifuged at ambienttemperature to obtain serum and processed for radioanalysis.

Two animals per time point were euthanized by intravenous injection ofeuthanasia solution followed by auscultation. The terminal time pointswere: 6 hours postdose, and 2, 7, 14, 21, and 28 days post-dose. Aqueoushumor, lens, vitreous humor, retina/choroid, and sclera were collectedfrom designated animals. The eyes were removed and any extraneous tissuewas trimmed from the outside of the eyeball. The aqueous humor wasremoved via syringe with a tuberculin needle and collected into apre-weighed gamma tube. The remaining eye was frozen in liquid nitrogenfor approximately 30 sec. On a chilled cutting surface, the cornea,iris, and lens were removed. The lens was rinsed with a small amount ofPBS and the rinse was collected into a gamma tube. The lens was blotteddry and placed in a pre-weighed gamma tube. The eye was re-frozen inliquid nitrogen as necessary. The sclera was cut and peeled back fromthe vitreous humor. The vitreous humor was removed and placed in apre-weighed container for solubilization. The sclera with theretina/choroid attached was dipped into a 1-ml rinse of PBS, which waspooled with the previous rinse, and the tissue was gently blotted dry.The retina/choroid was removed from the sclera and placed into separatepre-weighed gamma tubes. The appropriate surfaces and tools were wipedwith damp gauze and collected into a gamma tube for counting. Alltissues, rinses, and wipes were collected into a plastic container andprocessed for radioanalysis.

One eye per time point from select animals (including animals from thetime points 2, 14, and 28 days post-dose) was processed forradiochromatographic profiling of the vitreous humor. The right eye wasremoved and extraneous tissue was trimmed from the outside of theeyeball. The aqueous humor was removed via syringe with a tuberculinneedle and placed into a pre-weighed gamma tube. The maximum amount ofvitreous humor was collected using a 10-ml syringe with an 18-gaugeneedle. The contents were transferred to a pre-weighed container foranalysis by HPLC-Gamma-RAM™. Vitreous humor samples were keptrefrigerated or on wet ice for radiochromatographic profiling. Theremaining right eye tissue was placed into a pre-weighed container forsolubilization and processed for radioanalysis.

Single or duplicate aliquots of dosing formulations, serum, and wholeblood were mixed well and sampled for direct analysis of radioactivityvia the gamma counter. Ocular tissues were counted directly, diluted inPBS, or were solubilized in an incubating oven set at approximately 50°C. in a solution of 3N KOH/Methanol/TRITON™ X-100 and sampled intriplicate for analysis of radioactivity via the gamma counter. Allradioactive samples collected were counted for at least 5 min or 100,000counts in duplicate or triplicate, sample size allowing. All sampleresults (calculated at dpm/g sample) that had radioactivity greater than200 dpm were within 15% of the mean value.

Radiochromatographic profiling was performed on right eye vitreous humorsamples from one animal at 2, 14, and 28 hours post-dose. Samples wereanalyzed according to a fit-for-purpose method by HPLC-Gamma-RAM™. Peakareas and retention times were compared to assess test article integrityover time. To prepare the samples, a PRECELLYS® 24 homogenizer waspre-chilled to −10 to 0° C. with a cooled nitrogen stream. Zirconiabeads were added to an appropriate tube. With a positive placementpipette, a sample of vitreous humor was added to the tube containing theZirconia beads. The samples were placed in the pre-chilled PRECELLYS® 24homogenizer and homogenized at 6,500 RPM for six 60-sec cycles. Thetubes were centrifuged for 10 min at 14,000 RPM prior to analysis.

Pharmacokinetic parameters were estimated using PHOENIX® WinNonlin®version 6.2.1 (Certara USA, Inc., Princeton, N.J.). A non-compartmentalapproach consistent with the intravitreal route of administration wasused for parameter estimation. The half-life value forHA100K-rabFab-¹²⁵I in rabbit vitreous was 11.9 days, compared to 3.2days for rabFab from historical data (Shatz et al. Mol. Pharmaceutics2016; PubMed identifier (PMID) 27244474).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1-317. (canceled)
 318. A polynucleotide encoding an antibody thatspecifically binds vascular endothelial growth factor (VEGF), whereinthe antibody comprises the following six hypervariable regions (HVRs):(a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO:1); (b) an HVR-H2 comprising the amino acid sequence ofGX₁TPX₂GGX₃X₄X₅YX6DSVX₇X₈ (SEQ ID NO: 2), wherein X₁ is Ile or His, X₂is Ala or Arg, X₃ is Tyr or Lys, X₄ is Thr or Glu, X₅ is Arg, Tyr, Gln,or Glu, X₆ is Ala or Glu, X₇ is Lys or Glu, and X₈ is Gly or Glu; (c) anHVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3);(d) an HVR-L1 comprising the amino acid sequence of RASQX₁VSTAVA (SEQ IDNO: 4), wherein X₁ is Asp or Arg; (e) an HVR-L2 comprising the aminoacid sequence of X₁ASFLYS (SEQ ID NO: 5), wherein X₁ is Ser or Met; and(f) an HVR-L3 comprising the amino acid sequence of X₁QGYGX₂PFT (SEQ IDNO: 6), wherein X₁ is Gln, Asn, or Thr and X₂ is Ala, Gln, or Arg. 319.The polynucleotide of claim 318, wherein the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7), GITPAGGYEYYADSVKG (SEQ ID NO: 21),or GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10)or QQGYGNPFT (SEQ ID NO: 23).
 320. The polynucleotide of claim 319,wherein the antibody comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b) anHVR-H2 comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQ IDNO: 7); (c) an HVR-H3 comprising the amino acid sequence of FVFFLPYAMDY(SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acid sequence ofRASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising the amino acidsequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising theamino acid sequence of QQGYGAPFT (SEQ ID NO: 10).
 321. Thepolynucleotide of claim 320, wherein the antibody further comprises thefollowing heavy chain variable (VH) domain framework regions (FRs): (a)an FR-H1 comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) an FR-H3comprising the amino acid sequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR(SEQ ID NO: 15); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 16).
 322. The polynucleotide of claim 321,wherein the antibody further comprises the following light chainvariable (VL) domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).
 323. Apolynucleotide encoding an antibody that specifically binds VEGF,wherein the antibody comprises (a) a VH domain comprising an amino acidsequence having at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 11; (b) a VL domain comprising an amino acidsequence having at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 12; or (c) a VH domain as in (a) and a VL domainas in (b).
 324. The polynucleotide of claim 323, wherein the VH domainfurther comprises the following FRs: (a) an FR-H1 comprising the aminoacid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) anFR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO:14) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprising the aminoacid sequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and(d) an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ IDNO: 16).
 325. The polynucleotide of claim 324, wherein the VH domaincomprises the amino acid sequence of SEQ ID NO:
 11. 326. Thepolynucleotide of claim 323, wherein the VL domain further comprises thefollowing FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQID NO: 45); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), orGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20) orFGQGTKVEVK (SEQ ID NO: 55).
 327. The polynucleotide of claim 326,wherein the VL domain comprises the amino acid sequence of SEQ ID NO:12.
 328. A polynucleotide encoding an antibody that specifically bindsVEGF, wherein the antibody comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and (b) a VL domain comprising theamino acid sequence of SEQ ID NO:
 12. 329. A polynucleotide encoding anantibody that specifically binds VEGF, wherein the antibody comprises(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48and (b) a light chain comprising the amino acid sequence of SEQ ID NO:50.
 330. A polynucleotide encoding an antibody that specifically bindsVEGF, wherein the antibody comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 49 and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO:
 50. 331. The polynucleotide ofclaim 318, wherein the antibody is capable of inhibiting the binding ofVEGF to a VEGF receptor.
 332. The polynucleotide of claim 331, whereinthe VEGF receptor is VEGF receptor 1 (Flt-1) or VEGF receptor 2 (KDR).333. The polynucleotide of claim 318, wherein the antibody binds humanVEGF (hVEGF) with a Kd of about 2 nM or lower; has a melting temperature(Tm) of greater than about 83.5° C.; and/or has an isoelectric point(pI) of lower than
 8. 334. The polynucleotide of claim 318, wherein theantibody is monoclonal, human, humanized, or chimeric.
 335. Thepolynucleotide of claim 318, wherein the antibody is an antibodyfragment that binds VEGF.
 336. The polynucleotide of claim 335, whereinthe antibody fragment is selected from the group consisting of Fab,Fab-C, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments.
 337. The polynucleotideof claim 318, wherein the antibody is a monospecific antibody.
 338. Thepolynucleotide of claim 318, wherein the antibody is a multispecificantibody.
 339. The polynucleotide of claim 338, wherein themultispecific antibody is a bispecific antibody.
 340. A vectorcomprising the polynucleotide of claim
 318. 341. A host cell comprisingthe vector of claim
 340. 342. The host cell of claim 341, wherein thehost cell is prokaryotic.
 343. The host cell of claim 342, wherein thehost cell is Escherichia coli.
 344. The host cell of claim 341, whereinthe host cell is eukaryotic.
 345. The host cell of claim 344, whereinthe host cell is a 293 cell, a CHO cell, a yeast cell, or a plant cell.346. A method of producing an antibody that specifically binds to VEGF,the method comprising culturing a host cell that comprises the vector ofclaim 340 and recovering the antibody.
 347. The method of claim 346,wherein the host cell is prokaryotic.
 348. The method of claim 347,wherein the host cell is Escherichia coli.
 349. The method of claim 346,wherein the host cell is eukaryotic.
 350. The method of claim 349,wherein the host cell is a 293 cell, a CHO cell, a yeast cell, or aplant cell.